CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

BACKGROUND: Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. METHODS: Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. RESULTS: The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. CONCLUSIONS: A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders.

INTRODUCTION: Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are inherited red cell disorders caused by defects in various membrane proteins. The heterogeneous clinical presentation, biochemical and genetic abnormalities in HS and HE have been well documented. The need to raise the awareness of HSt, albeit its much lower prevalence than HS, is due to the undesirable outcome of splenectomy in these patients. METHODS: The scope of this guideline is to identify the characteristic clinical features, the red cell parameters (including red cell morphology) for these red cell disorders associated, respectively, with defective cytoskeleton (HS and HE) and abnormal cation permeability in the lipid bilayer (HSt) of the red cell. The current screening tests for HS are described, and their limitations are highlighted. RESULTS: An appropriate diagnosis can often be made when the screening test result(s) is reviewed together with the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, monovalent cation flux measurement, and molecular analysis of membrane protein genes are specialist tests for further investigation. CONCLUSION: Specialist tests provide additional evidence in supporting the diagnosis and that will facilitate the management of the patient. In the case of a patient's clinical phenotype being more severe than the affected members within the immediate family, molecular testing of all family members is useful for confirming the diagnosis and allows an insight into the molecular basis of the abnormality such as a recessive mode of inheritance or a de novo mutation.

Revisiting the concept of the 'problem young driver' within the context of the 'young driver problem': who are they?

For decades there have been two young driver concepts: the 'young driver problem' where the driver cohort represents a key problem for road safety; and the 'problem young driver' where a sub-sample of drivers represents the greatest road safety problem. Given difficulties associated with identifying and then modifying the behaviour of the latter group, broad countermeasures such as graduated driver licensing (GDL) have generally been relied upon to address the young driver problem. GDL evaluations reveal general road safety benefits for young drivers, yet they continue to be overrepresented in fatality and injury statistics. Therefore it is timely for researchers to revisit the 'problem young driver' concept to assess its potential countermeasure implications. This is particularly relevant within the context of broader countermeasures that have been designed to address the 'young driver problem' Personal characteristics, behaviours and attitudes of 378 Queensland novice drivers aged 17-25 years were explored during their pre-, Learner and Provisional 1 (intermediate) licence as part of a larger longitudinal project. Self-reported risky driving was measured by the Behaviour of Young Novice Drivers Scale (BYNDS), and five subscale scores were used to cluster the drivers into three groups (high risk n=49, medium risk n=163, low risk n=166). High risk 'problem young drivers' were characterised by greater self-reported pre-Licence driving, unsupervised Learner driving, and speeding, driving errors, risky driving exposure, crash involvement, and offence detection during the Provisional period. Medium risk drivers were also characterised by more risky road use than the low risk group. Interestingly problem young drivers appear to have some insight into their high-risk driving, since they report significantly greater intentions to bend road rules in future driving. The results suggest that tailored intervention efforts may need to target problem young drivers within the context of broad countermeasures such as GDL which address the young driver problem in general. Experiences such as crash-involvement could be used to identify these drivers as a preintervention screening measure.

Hereditary red cell membrane disorders and laboratory diagnostic testing.

This overview describes two groups of nonimmune hereditary hemolytic anemias caused by defects in membrane proteins located in distinct layers of the red cell membrane. Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) represent disorders of the red cell cytoskeleton. Hereditary stomatocytoses represents disorders of cation permeability in the red cell membrane. The current laboratory screening tests for HS are the osmotic fragility test, acid glycerol lysis time test (AGLT), cryohemolysis test, and eosin-5'-maleimide (EMA)-binding test. For atypical HS, SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is carried out to confirm the diagnosis. The diagnosis of HE/HPP is based on abnormal red cell morphology and the detection of protein 4.1R deficiency or spectrin variants using gel electrophoresis. None of screening tests can detect all HS cases. Some testing centers (a survey of 25 laboratories) use a combination of tests (e.g., AGLT and EMA). No specific screening test for hereditary stomatocytoses is available. The preliminary diagnosis is based on presenting a compensated hemolytic anemia, macrocytosis, and a temperature or time dependent pseudohyperkalemia in some patients. Both the EMA-binding test and the osmotic fragility test may help in differential diagnosis of HS and hereditary stomatocytosis.

Confirmatory factor analysis of the Behaviour of Young Novice Drivers Scale (BYNDS).

PURPOSE: The greatly increased risk of being killed or injured in a car crash for the young novice driver has been recognised in the road safety and injury prevention literature for decades. Risky driving behaviour has consistently been found to contribute to traffic crashes. Researchers have devised a number of instruments to measure this risky driving behaviour. One tool developed specifically to measure the risky behaviour of young novice drivers is the Behaviour of Young Novice Drivers Scale (BYNDS) (Scott-Parker et al., 2010). The BYNDS consists of 44 items comprising five subscales for transient violations, fixed violations, misjudgement, risky driving exposure, and driving in response to their mood. The factor structure of the BYNDS has not been examined since its development in a matched sample of 476 novice drivers aged 17-25 years. METHOD: The current research attempted to refine the BYNDS and explore its relationship with the self-reported crash and offence involvement and driving intentions of 390 drivers aged 17-25 years (M=18.23, SD=1.58) in Queensland, Australia, during their first 6 months of independent driving with a Provisional (intermediate) driver's licence. A confirmatory factor analysis was undertaken examining the fit of the originally proposed BYNDS measurement model. RESULTS: The model was not a good fit to the data. A number of iterations removed items with low factor loadings, resulting in a 36-item revised BYNDS which was a good fit to the data. The revised BYNDS was highly internally consistent. Crashes were associated with fixed violations, risky driving exposure, and misjudgement; offences were moderately associated with risky driving exposure and transient violations; and road-rule compliance intentions were highly associated with transient violations. CONCLUSIONS: Applications of the BYNDS in other young novice driver populations will further explore the factor structure of both the original and revised BYNDS. The relationships between BYNDS subscales and self-reported risky behaviour and attitudes can also inform countermeasure development, such as targeting young novice driver non-compliance through enforcement and education initiatives.

Fatalism and its implications for risky road use and receptiveness to safety messages: a qualitative investigation in Pakistan.

Given the increasing vehicle numbers and expanding road construction in developing countries, the importance of safe road user behaviour is critical. Road traffic crashes (RTCs) are a significant problem in Pakistan; however, the factors that contribute to RTCs in Pakistan are not well researched. Fatalistic beliefs are a potential barrier to the enhancement of road safety, especially participation in health-promoting and injury prevention behaviours, and also contribute to risk taking. Fatalistic beliefs relating to road safety have been found in some developing countries, although research is scarce and indicates that the nature and extent of fatalism differs in each country. Qualitative research was undertaken with a range of drivers, religious orators, police and policy makers to explore associations between fatalism, risky road use and associated issues. Findings indicate that fatalistic beliefs are pervasive in Pakistan, are strongly linked with religion, present a likely barrier to road safety messages and contribute to risky road use. Fatalism appears to be a default attribution of RTC and the intensity of belief in fate surpasses the kinds of fatalism noted in the limited existing literature. These findings have importance to developing road safety countermeasures in countries where fatalistic beliefs are strong.

Young and unlicensed: risky driving before entering the licensing system.

OBJECTIVE: On-road driving before gaining a valid license (prelicense driving) represents a risk for all road users. Prelicense driving among young people who obtained a provisional license within an enhanced graduated driver licensing program in Queensland, Australia, was investigated. METHODS: Recently licensed drivers (n = 1032) aged 17 to 19 years (M = 17.54) completed a survey exploring their driving experiences while on their learner's license. Six months later, 355 of these drivers completed the same survey exploring their experiences on their provisional (intermediate) license. RESULTS: Twelve percent of participants reported prelicense driving. Prelicense drivers reported significantly more risky driving as learners and provisional drivers. CONCLUSIONS: Prelicense drivers not only place themselves and other road users at risk at the time but also continue to do so through their subsequent risky driving. Prelicense driving should be discouraged, and parents should be encouraged to monitor car use and the driving behavior of their children.

Optimism about safety and group-serving interpretations of safety among pedestrians and cyclists in relation to road use in general and under low light conditions.

Drivers are known to be optimistic about their risk of crash involvement, believing that they are less likely to be involved in a crash than other drivers. However, little comparative research has been conducted among other road users. In addition, optimism about crash risk is conceptualised as applying only to an individual's assessment of his or her personal risk of crash involvement. The possibility that the self-serving nature of optimism about safety might be generalised to the group-level as a cyclist or a pedestrian, i.e., becoming group-serving rather than self-serving, has been overlooked in relation to road safety. This study analysed a subset of data collected as part of a larger research project on the visibility of pedestrians, cyclists and road workers, focusing on a set of questionnaire items administered to 406 pedestrians, 838 cyclists and 622 drivers. The items related to safety in various scenarios involving drivers, pedestrians and cyclists, allowing predictions to be derived about group differences in agreement with items based on the assumption that the results would exhibit group-serving bias. Analysis of the responses indicated that specific hypotheses about group-serving interpretations of safety and responsibility were supported in 22 of the 26 comparisons. When the nine comparisons relevant to low lighting conditions were considered separately, seven were found to be supported. The findings of the research have implications for public education and for the likely acceptance of messages which are inconsistent with current assumptions and expectations of pedestrians and cyclists. They also suggest that research into group-serving interpretations of safety, even for temporary roles rather than enduring groups, could be fruitful. Further, there is an implication that gains in safety can be made by better educating road users about the limitations of their visibility and the ramifications of this for their own road safety, particularly in low light.

Mileage, car ownership, experience of punishment avoidance, and the risky driving of young drivers.

OBJECTIVE: Young drivers are at greatest risk of injury or death from a car crash in the first 6 months of independent driving. In Queensland, the graduated driver licensing (GDL) program was extensively modified in July 2007 in order to reduce this risk. Increased mileage and car ownership have been found to play a role in risky driving, offenses, and crashes; however, GDL programs typically do not consider these variables. In addition, young novice drivers' experiences of punishment avoidance have not previously been examined. This article explores the mileage (duration and distance), car ownership, and punishment avoidance behaviors of young newly licensed intermediate (provisional) drivers and their relationship to risky driving, crashes, and offenses. METHODS: Drivers (n = 1032) aged 17 to 19 years recruited from across Queensland for longitudinal research completed survey 1 exploring prelicense and learner experiences and sociodemographic characteristics. survey 2 explored the same variables with a subset of these drivers (n = 341) after they had completed their first 6 months of independent driving. RESULTS: Most young drivers in survey 2 reported owning a vehicle and paying attention to police presence. Drivers who had their own cars reported significantly greater mileage and more risky driving. Novices who drove more kilometers, spent more hours each week driving, or avoided actual and anticipated police presence were more likely to report risky driving. These drivers were also more likely to report being detected by police for a driving-related offense. The media, parents, friends, and other drivers play a pivotal role in informing novices of on-road police enforcement operations. CONCLUSIONS: GDL programs should incorporate education for the parent and novice driver regarding the increased risks associated with greater driving, particularly when the novice driver owns a vehicle. Parents should be encouraged to delay exclusive access to a vehicle. Parents should also consider whether their young novices will deliberately avoid police if they are aware of their location. This may reinforce not only the risky behavior but also young novices' beliefs that their parents condone this behavior.

A genomic deletion causes truncation of α-spectrin and ellipto-poikilocytosis.

We report on a truncated α-spectrin chain, spectrin(Exeter), associated with ellipto-poikilocytosis. Analysis of erythrocyte membranes of affected individuals revealed a truncated α-spectrin chain with normal amounts of spectrin dimer. In the proband and her father, one haploid set of α-spectrin cDNA lacked exons 11 and 12, leading to partial deletion of repeats α4 and α5 (83 amino acids) of the α-spectrin chain. In one allele of genomic DNA, a 3567bp deletion starting in intron 10 and ending in intron 12 of the SPTA1 gene was found. The common polymorphic SPTA1 α(LELY) allele was found in trans to the SPTA1αExeter allele in the proband. The proband had inherited the SPTA1Exeter allele from her father and the αLELY allele from her healthy, asymptomatic mother. This is the first report of an interstitial deletion in the SPTA1 gene associated with ellipto-poikilocytosis.

Detection of hereditary pyropoikilocytosis by the eosin-5-maleimide (EMA)-binding test is attributable to a marked reduction in EMA-reactive transmembrane proteins.

INTRODUCTION: Hereditary spherocytosis (HS) and hereditary pyropoikilocytosis (HPP, severe form of hereditary elliptocytosis) are unrelated red cell disorders caused by defects in distinct regions of the red cell cytoskeleton. The high predictive value of the eosin-5-maleimide (EMA)-binding test for the diagnosis of HS is because of its interaction with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47, which are reduced on HS red cells. Our study was undertaken to determine why EMA-labelled HPP red cells were previously found to give much lower fluorescence readings than HS. METHODS: Flow cytometry was used to determine the relative amounts of monoclonal antibodies bound to red cells from normal adults, HS and HPP groups. Confocal microscopy was used to visualise the overall staining pattern of the red cells with selected antibodies. RESULTS: In flow cytometry, HPP red cells gave lower antibody binding to the four EMA-reactive membrane proteins than HS red cells and bound less antibody to glycophorins A and C, and CD59. Confocal images of Rh protein and band 3 immunostaining revealed a greater number of HPP red cells having partial or no fluorescence than in HS and normal controls. CONCLUSION: Lesser amounts of EMA-reactive membrane proteins were detected in HPP than HS red cells, thus confirming their lower fluorescence readings in the EMA-binding test. The concomitant reduction in glycophorins A and C, and CD59 in HPP could have caused cellular contraction, resulting in poikilocytosis.

Vitrification assays with embryos from a cold tolerant sub-arctic fish species.

Pseudopleuronectes americanus is a Northern teleost species that produces antifreeze proteins (AFPs) to protect them from freezing during the winter. These AFPs bind to ice crystals to inhibit their growth, and they also protect cell membranes at low temperatures. In this study, vitrification trials were done with fish embryos at three different developmental stages, using two different protocols for incorporating the vitrifying solutions. Toxicity of the cryoprotectants and permeability to dimethyl sulfoxide were analyzed. Embryos were vitrified in 0.5 ml straws by direct immersion in liquid nitrogen, and their morphology and development analyzed following thaw. The embryos responded well to vitrification as evidenced by the high percentage that exhibited good morphology following thaw. Although none of the embryos hatched, a small percentage (0.92%) of them showed active movements within the chorion and continued to develop for a number of days following thaw. This is the first record of post-thaw development of vitrified fish embryos.

Guidelines for the diagnosis and management of hereditary spherocytosis.

Hereditary spherocytosis (HS) is a heterogeneous group of disorders with regard to clinical severity, protein defects and mode of inheritance. It is relatively common in Caucasian populations; most affected individuals have mild or only moderate haemolysis. There is usually a family history, and a typical clinical and laboratory picture so that the diagnosis is often easily made without additional laboratory tests. Atypical cases may require measurement of erythrocyte membrane proteins to clarify the nature of the membrane disorder and in the absence of a family history, occasionally molecular genetic analysis will help to determine whether inheritance is recessive or non-dominant. It is particularly important to rule out stomatocytosis where splenectomy is contraindicated because of the thrombotic risk. Mild HS can be managed without folate supplements and does not require splenectomy. Moderately and severely affected individuals are likely to benefit from splenectomy, which should be performed after the age of 6 years and with appropriate counselling about the infection risk. In all cases careful dialogue between doctor, patient and the family is essential. Laparoscopic surgery, when performed by experienced surgeons, can result in a shorter hospital stay and less pain.

Reconstruction of the trachea with a tubed radial forearm free flap.

BACKGROUND: We present the case of a patient with adenoid cystic carcinoma of the trachea who had 60 mm of the trachea excised and reconstructed with a stented radial forearm free flap. The patient was well in the immediate postoperative period with good function of the neotrachea. Problems developing after the reconstruction included proximal stricture, sputum retention, and recurrent pneumonia. RESULT: The patient died of malignant hypercalcemia 16 months after the reconstruction. To our knowledge this is the first reported case of a total tracheal resection and reconstruction with a combination of free tissue transfer and internal stenting. CONCLUSION: We conclude that tracheal reconstruction has the potential to provide a reliable airway in patients not able to be reconstructed with a primary anastomosis.