Using expert-elicitation to deliver biodiversity monitoring priorities on a Mediterranean island.

Biodiversity monitoring plays an essential role in tracking changes in ecosystems, species distributions and abundances across the globe. Data collected through both structured and unstructured biodiversity recording can inform conservation measures designed to reduce, prevent, and reverse declines in valued biodiversity of many types. However, given that resources for biodiversity monitoring are limited, it is important that funding bodies prioritise investments relative to the requirements in any given region. We addressed this prioritisation requirement for a biodiverse Mediterranean island (Cyprus) using a three-stage process of expert-elicitation. This resulted in a structured list of twenty biodiversity monitoring needs; specifically, a hierarchy of three groups of these needs was created using a consensus approach. The most highly prioritised biodiversity monitoring needs were those related to the development of robust survey methodologies, and those ensuring that sufficiently skilled citizens are available to contribute. We discuss ways that the results of our expert-elicitation process could be used to support current and future biodiversity monitoring in Cyprus.

Compliance with the European trauma guidelines: An observational single centre study.

OBJECTIVES: The European trauma guidelines were developed to assist clinicians in the early phase of trauma management to diagnose and treat coagulopathy and bleeding. This study aimed to determine compliance with these European trauma guidelines in a French referral trauma centre. METHODS: Medical charts of trauma patients with an injury severity score≥16 admitted between January 2013 and December 2014 were reviewed. Compliance with 21 recommendations in the first 24-hours of patient management was assessed. RESULTS: There were 145 patients with median ISS of 34 [IQR 25-41]. A good level of compliance (i.e. applied in≥80% of patients) was identified for nine recommendations, inconsistent compliance (i.e. applied in 50 to 79% of patients) for six recommendations, including fibrinogen levels at hospital admission and achievement of a target mean arterial blood pressure (MAP)>80mmHg in patients with major bleeding and TBI (55.5%), and poor compliance (i.e. applied in<50% of patients) for another six recommendations. Poorly applied recommendations included early measurement of lactate or base deficit (32%), early administration of tranexamic acid (18%), and achievement of normocapnia in patients with TBI undergoing invasive ventilation (3%). CONCLUSIONS: In a referral trauma centre, nine of the 21 evaluable recommendations in the European trauma guidelines were applied in≥80% of patients. Early diagnosis and treatment of trauma-related coagulopathy was identified as an area for significant practice improvement. In patients with TBI, efforts should be made to achieve the targeted MAP and to maintain normocapnia.

How clinicians can minimize transfusion-related adverse events?

OBJECTIVES: Transfusion-related adverse events (TRAE) can contribute to patient morbidity and mortality. In this brief narrative review, the strategies that clinicians can apply at the bedside to avoid TRAE are discussed. METHODS: Strategies to avoid the following five types of TRAE were reviewed: transfusion-associated circulatory overload (TACO), transfusion-related acute lung injury (TRALI), transfusion-associated hypothermia (TAH), transfusion-related allergic reactions (TRAR) and acute haemolytic transfusion reactions (AHTR). RESULTS: Minimizing exposure to blood components is fundamental to TRAE avoidance. Pre-transfusion assessment can identify patients at risk of TACO, TRAR and TAH, and avoidance steps implemented. Preventive strategies for TACO include lower transfusion rate, 'one unit at a time' transfusion policy and possibly diuretic medication. Patients with past history of TRAR should preferably be given plasma-free blood components; anti-histamine medication prior to transfusion could be considered. TAH is common in the massive transfusion setting, particularly trauma patients. Warming of patients are key strategies to avoid TAH. Identification of patients at risk of TRALI is more opaque; however, any measures that limit pulmonary inflammation prior to transfusion may decrease the risk of TRALI. Causes of AHTR are commonly due to human error and failure to apply rigorous cross-checks of patient and issued RBC component blood groups. CONCLUSIONS: Beneficial strategies to avoid TRAE include judicious use of blood components, identification of high-risk patients, adherence to recommended clinical processes and awareness of TRAE pathophysiology. More evidence is warranted to better guide clinicians in the prevention of TRAE.

Longer storage of red blood cells is associated with increased in vitro erythrophagocytosis.

BACKGROUND AND OBJECTIVES: Refrigerated storage of red blood cells (RBCs) induces numerous changes that may target the cells for erythrophagocytosis following transfusion. The influence of storage upon the phagocytosis of unseparated and fractionated young and old stored RBCs was investigated using two in vitro quantitative phagocytosis assays. MATERIALS AND METHODS: Leucocyte-depleted RBC units were sampled at day 1 or 42 of storage. Young and old RBCs were fractionated at day 1 by density centrifugation and stored in paediatric packs for up to 42 days. RBCs were labelled with the fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1. Erythrophagocytosis was quantified by flow cytometry and plate fluorometric assays. RESULTS: A higher proportion of THP-1 cells phagocytosed RBCs stored for 42 days compared with 1 day (41% and 24% respectively; P<0·0001). This was associated with an increased mean number of RBCs phagocytosed per THP-1 cell (5·2±0·6 and 3·3±0·2 respectively; P<0·002). Erythrophagocytosis of fractionated young and old RBCs increased with longer storage duration up to 28 days (P<0·05). However, no significant differences were observed between erythrophagocytosis of young and old RBCs. CONCLUSION: The susceptibility of stored RBCs to erythrophagocytosis significantly increased with longer storage time of the RBC units. Storage duration of RBCs had a greater influence on in vitro erythrophagocytosis than the chronological age of the RBCs at donation.

Right of the living dead? Consent to experimental surgery in the event of cortical death.

Ravelingien et al have suggested that early human xenotransplantation trials should be carried out on patients who are in a permanent vegetative state (PVS) and who have previously granted their consent to the use of their bodies in such research in the event of their cortical death. Unfortunately, their philosophical defence of this suggestion is unsatisfactory in its current formulation, as it equivocates on the key question of the status of patients who are in a PVS. The solution proposed by them rests on the idea that it should be up to people themselves to determine when they should be treated as dead. Yet the authors clearly believe (and state) that patients who are in a PVS are in fact dead. Finally, given the public good that their proposal is intended to achieve, the moral importance they place on the consent of a person to the use of his or her body in research is ultimately only defensible in so far as this consent represents the wishes of a living person. It is thus only a gentle caricature of their position to suggest that according to their account, consent to participation in xenotransplantation research is a "right of the living dead". The equivocation by Ravelingien et al on the question of whether these people are living or dead means that they avoid confronting the implications of their argument. The solution proposed by Ravelingien et al to the problem of how we should proceed with xenotransplantation research is therefore not as neat as it first seems to be.

Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression.

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.

Supernatant from stored red blood cell primes inflammatory cells: influence of prestorage white cell reduction.

BACKGROUND: The contribution of RBC transfusion to adverse patient outcomes is controversial. There is conflicting clinical data and limited biologic data that provide an underpinning biologic rationale for any adverse impacts from RBC transfusion. This study used in-vitro measures of PMN stimulation to determine the ability of supernatant from RBCs to stimulate allogeneic WBCs and to determine the influence of residual donor WBCs and storage time on the proinflammatory potential of RBCs. STUDY DESIGN AND METHODS: Three types of RBCs were assessed: standard non-WBC-reduced RBCs (S-RBCs), buffy coat-poor RBCs (BCP-RBCs), and prestorage WBC-filtered RBC (LF-RBCs). Supernatant was collected weekly up to Day 42 of storage. PMN priming by supernatant from RBCs was determined by three methods: induction of CD11b expression on PMNs, induction of IL-8 release from PMNs, and the chemotactic effect of supernatant on PMNs. RESULTS: Supernatant from S-RBCs induced the expression of CD11b on PMNs, primed PMNs to release IL-8, and was chemotactic for PMNs. The magnitude of this PMN-priming progressively amplified with storage time. In contrast, supernatant from BCP-RBCs or LF-RBCs did not significantly prime PMNs. The PMN-priming effect of supernatant from RBCs correlated more closely with the level of MNCs in the RBCs than PMN content. CONCLUSION: Supernatant from stored S-RBCs prime unstimulated allogeneic PMNs in vitro. Prestorage buffy-coat WBC reduction was as effective as WBC depletion in abrogating this proinflammatory response elicited by supernatants from RBCs. The clinical consequences, if any, of these findings for transfusion recipients are unknown.

Microbial screening of UC blood units by an automated culture system: effect of delayed testing on bacterial detection.

BACKGROUND: Microbial screening is a mandatory test for banked UC blood (UCB) to comply with the code of good manufacturing practice (GMP). Cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing. This study investigated the influence of >/=24 h delays in initiating automated microbial screening on the detection of bacteria in UCB, by analysis of specimens deliberately spiked with bacteria and the recovery of bacteria from cryopreserved spiked-UCB. MATERIALS AND METHODS: UCB was processed according to standard CBB procedures and spiked with Staphylococcus epidermidis or Escherichia coli [2-2000 colony forming units (CFU)/mL]. Spiked-UCB (0.5 mL) was (1) held at room temperature (RT) and inoculated into pediatric BacT/Alert bottles (bioMérieux) at Days 1, 4 and 7 (delayed inoculation); and (2) inoculated directly (Day 0) into replicate BacT/Alert bottles and held at RT for 1, 4 or 7 days before loading onto the BacT/ALERT system (delayed loading). Spiked-UCB samples were cryopreserved. Bacterial counts were quantitated on horse blood agar plates. RESULTS: Bacterial growth in UCB spiked with a single bacterium was capable of detection by the BacT/ALERT system. S. epidermidis grew in all conditions of delayed testing (ie. delayed inoculation and delayed loading). E. coli failed to grow under conditions of delayed inoculation but grew at all time points of delayed loading. S. epidermidis and E. coli were recovered from cryopreserved spiked-UCB. DISCUSSION: Inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB.

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy.

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.

The short tandem repeat locus VWF2 in Intron 40 of the von Willebrand factor gene consists of two polymorphic sub-loci.

This study describes the complex nucleotide sequence structure of the TCTA short tandem repeat (STR) locus, VWF2. Eight alleles of VWF2 were observed in a population of 116 unrelated Caucasian individuals. The alleles ranged in size from 150 to 178 base pairs (bp). Sequence analysis of the isolated alleles revealed two polymorphic regions that were named sub-loci VWF2-a and VWF2-b. VWF2-a is located at the 5' end of the conventional locus, whilst VWF2-b is located at the 3' end. The two sub-loci are joined by a 30-nucleotide non-polymorphic sequence which contains two additional TCTA motif repeats. A semi-nested polymerase chain reaction (PCR) was designed to amplify the VWF2-b region in conjunction with the standard VWF2 amplification. This new amplification method enabled a higher level of allele discrimination than could be achieved by assigning alleles according to size. A cohort of 99 unrelated individuals was tested with this method. VWF2-a expressed five different alleles ranging from zero motif repeats to four motif repeats, while VWF2-b alleles ranged from 8 to 14 motif repeats. Allelic configuration based on the VWF2-a and VWF2-b sub-alleles revealed 23 unique configurations out of a possible 31 for the original eight VWF2 alleles. In conclusion, the VWF2 is a highly polymorphic STR locus with potential application for forensic and parentage testing.

Does the site of intestinal delivery of oleic acid alter the ileal brake response?

Previous work has demonstrated that high doses of oleic acid can activate the ileal brake but the importance of site of delivery has yet to be investigated. The objective of this study was to use modified release capsules to release oleic acid in different regions of the intestine. When tested by in vitro dissolution in pH 6.8 phosphate buffer, one batch released the contents almost immediately, another after around 30 min and the last batch after around 60-70 min. The effect of oleic acid release site on the ileal brake was assessed by the measurement of transit time of radiolabelled non disintegrating tablets by gamma scintigraphy. The results demonstrated that the transit of tablets could be slowed down by oleic acid and therefore it appears the ileal brake can be activated along the entirety of the small intestine.

Topographical distribution of blood supply to the anal canal.

BACKGROUND: It has been suggested that anal fissure is an ischaemic ulcer caused by a combination of poor blood supply to the posterior midline of the anal canal and spasm of the internal anal sphincter. This study investigated the topographical distribution of blood supply to quadrants of the anal canal above and below the dentate line. METHODS: Cadaveric anal canals were removed and 1-cm blocks were cut above and below the dentate line. Blocks were sectioned at 10 microm and every 25th section was mounted. Using the technique of systematic random sampling, fields in the subanodermal space and the internal anal sphincter in posterior, lateral and anterior quadrants of the anal canal were chosen. The numbers of small arterioles in each field were counted. Mean counts were compared for both subanodermal space and internal anal sphincter between quadrants and levels above and below the dentate line using Page's L test for trends. RESULTS: Anal canals from eight cadavers were examined. There was a significant trend to an increasing number of arterioles from posterior to anterior in the subanodermal space at all levels and at two of three levels in the internal anal sphincter. CONCLUSION: The arteriolar density is less in the posterior quadrant throughout the anal canal. It may be that this poor blood supply predisposes to the development of anal fissures at their most common site in the posterior midline.

The effect of oleic acid on the human ileal brake and its implications for small intestinal transit of tablet formulations.

PURPOSE: A human volunteer study was carried out to investigate whether activation of the ileal brake mechanism affects the transit of tablets through the small intestine. METHODS: Oleic acid, which has previously been shown to activate the brake, was delivered to the small intestine in a modified release capsule at doses of 300 mg, 600 mg and 1200 mg. The effect of the oleic acid was determined by measuring the transit of two sets of radiolabelled tablets by gamma scintigraphy. One set of tablets was dosed with the capsule and the other one hour later. RESULTS: The results show that in the majority of the volunteers small intestinal residence time was greater with the oleic acid than control. The effect was most pronounced in the tablets given concomitantly with the capsule and with the higher doses of oleic acid. CONCLUSIONS: The ileal brake, activated by oleic acid, can slow the transit of tablets through the small intestine.

Serologic and molecular investigations of a chimera.

A chimeric individual possesses two or more genetically distinct cell populations. Although the chimerism may not be evident in all gene systems, various loci display greater numbers of alleles than genetically "normal" individuals. The proposita was referred for further laboratory investigation due to a mixed-field ABO blood group reaction following routine antenatal testing. Various molecular (HLA class II, ABO genotyping, and 10 short tandem repeat [STR] microsatellites) and serologic (HLA class I and red cell blood groups) typing techniques were employed to investigate a number of polymorphic loci located on different chromosomes. Chimerism was identified in 8 out of the 14 chromosomes tested: chromosome 1 (Duffy), 6 (HLA class I and II), 9 (ABO), 11 (HUMTH01), 12 (HUMPLA2A1), 15 (HUMFES/FPS), 18 (Kidd) and 21 (D21S11). The proposita was determined to be a probable dispermic chimera, based on the results of the serology and molecular studies.

Are ophthalmic surgeons aware that starch powdered surgical gloves are a risk factor in ocular surgery?

PURPOSE: To assess the level of awareness among UK ophthalmic surgeons of the potential risks from starch powdered surgical gloves during ophthalmic surgery and to show by electron microscopy that starch granule contamination can occur during ophthalmic surgery. SETTING: A sample (N = 46) of UK ophthalmologists from the North of England, UK. METHODS: Type of glove usage and awareness of the possible risks from starch powdered surgical gloves were assessed by means of a questionnaire sent to ophthalmic surgeons in the North of England. The surface of a polymethylmethacrylate (PMMA) intraocular lens (IOL) handled with a starch powdered surgical glove was examined by electron microscopy for evidence of starch contamination. RESULTS: Of the sampled ophthalmic surgeons (46), 89.1% considered it important to use starch free surgical gloves and the 84.8% already did so. Starch granule contamination was seen by electron microscopy on the surface of a PMMA IOL which had been handled with starch powdered surgical gloves. CONCLUSIONS: Although there has been sporadic attention in the ophthalmic literature to the risks associated with starch powdered surgical gloves in ophthalmology, up to 15% of UK ophthalmic surgeons may still be using starch powdered gloves. The authors show that starch powder contamination of ophthalmic materials can actually occur and remind ophthalmologists that this has been reported in the literature as a possible cause of sterile intra and extraocular inflammation.