BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

Calcium cycling is integral to muscle performance during the rapid muscle contraction and relaxation of high-intensity exercise. Ca(2+) handling is altered by diabetes mellitus, but has not previously been investigated in human skeletal muscle. We investigated effects of high-intensity exercise and sprint training on skeletal muscle Ca(2+) regulation among men and women with type 1 diabetes (T1D, n = 8, 3F, 5M) and matched non-diabetic controls (CON, n = 8, 3F, 5M). Secondarily, we examined sex differences in Ca(2+) regulation. Subjects undertook 7 weeks of three times-weekly cycle sprint training. Before and after training, performance was measured, and blood and muscle were sampled at rest and after high-intensity exercise. In T1D, higher Ca(2+)-ATPase activity (+28%) and Ca(2+) uptake (+21%) than in CON were evident across both times and days (P < 0.05), but performance was similar. In T1D, resting Ca(2+)-ATPase activity correlated with work performed until exhaustion (r = 0.7, P < 0.01). Ca(2+)-ATPase activity, but not Ca(2+) uptake, was lower (-24%, P < 0.05) among the women across both times and days. Intense exercise did not alter Ca(2+)-ATPase activity in T1D or CON. However, sex differences were evident: Ca(2+)-ATPase was reduced with exercise among men but increased among women across both days (time × sex interaction, P < 0.05). Sprint training reduced Ca(2+)-ATPase (-8%, P < 0.05), but not Ca(2+) uptake, in T1D and CON. In summary, skeletal muscle Ca(2+) resequestration capacity was increased in T1D, but performance was not greater than CON. Sprint training reduced Ca(2+)-ATPase in T1D and CON. Sex differences in Ca(2+)-ATPase activity were evident and may be linked with fibre type proportion differences.

Validation of an in vitro contractility assay using canine ventricular myocytes.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ~36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration-effect curves were constructed for each compound in 4-30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6-8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds.

British Society for Histocompatibility & Immunogenetics and British Transplantation Society guidelines for the detection and characterisation of clinically relevant antibodies in allotransplantation.

Ongoing technological developments in antibody detection and characterisation allowing relative quantitation of HLA-specific antibody levels, combined with crossmatch results, now allow a graded assessment of patient potential donor immunological risk for allotransplantation, rather than a simple 'positive' or 'negative' categorization of crossmatch results. These developments have driven a thorough revision of the British Society for Histocompatibility & Immunogenetics and British Transplantation Society Guidelines for the Detection and Characterisation of Clinically Relevant Antibodies in Allotransplantation. These newly published revised Guidelines contain a number of recommendations as to best practice for antibody detection and crossmatching for the transplantation of a wide range of solid organs and tissues. These recommendations are briefly summarized in this article.

Glucose tolerance and physical activity level in people with spinal cord injury.

STUDY DESIGN: Cross-sectional, observational study. OBJECTIVES: To evaluate the associations of physical activity and neurological lesion level with glucose tolerance in people with spinal cord injury (SCI). SETTING: New South Wales, Australia. METHODS: Twenty-five people (5 women, 20 men) with SCI (>6 months post-injury) aged between 18 and 65 years were recruited. Exclusion criteria included known coronary heart disease, stroke or diabetes. Participants underwent an oral glucose tolerance test. Fasting and 2-h plasma glucose concentrations were classified according to the World Health Organization categories of glycemia. Participants also completed the Physical Activity Scale for Individuals with Physical Disabilities and mean MET-hours day(-1) was calculated. Associations with the 2-h plasma glucose concentration were calculated through multiple and stepwise regressions. RESULTS: Participants presented with complete or incomplete tetraplegia (n=11 TETRA) or complete or incomplete paraplegia (n=14 PARA) with neurological lesion levels ranging from C3/4 to T12. Mean 2-h plasma glucose was 7.13+/-2.32 mmol l(-1). Nine participants had disordered glycemia (n=6 TETRA; n=3 PARA) and the remaining participants had normal glucose tolerance. Those participants with normal glucose tolerance participated in more moderate-vigorous and strength exercise and undertook more non-exercise-related mobility than those with disordered glycemia. Physical activity and age, but not lesion level were independent determinants of 2-h plasma glucose concentration (r=0.683, P=0.001), explaining 47% of the variance. CONCLUSION: Physical activity level is independently associated with glucose tolerance in people with SCI. Non-exercise activity may also be important for maintaining normal glycemia.

De novo membranous nephropathy associated with donor-specific alloantibody.

Recent evidence suggests that alloantibody may play an aetiological role in the pathogenesis of membranous glomerulopathy in native kidneys. There is an increased awareness of the significance of alloantibody on renal transplant outcome, particularly with the development of more sensitive assays. We describe a kidney transplant patient who developed de novo membranous glomerulopathy (DNMG) with heavy proteinuria in the context of a donor-specific alloantibody (DSA) directed against HLA DQ7. Proteinuria resolved and kidney function stabilized following treatment with mycophenolate mofetil and an angiotensin receptor blocker. The titre of the DSA fell in parallel with resolution of the proteinuria. This is the first reported case of DNMG after kidney transplantation clearly associated with a DSA. We hypothesize that de novo membranous glomerulopathy may be an atypical manifestation of acute antibody-mediated damage. Cases of DNMG should be screened for alloantibody and the presence of alloantibody may influence the choice of therapy.

Fitness training for cardiorespiratory conditioning after traumatic brain injury.

BACKGROUND: Cardiorespiratory deconditioning is a common sequelae after traumatic brain injury (TBI). Clinically, fitness training is implemented to address this impairment, however this intervention has not been subject to rigorous review. OBJECTIVES: The primary objective was to evaluate whether fitness training improves cardiorespiratory fitness in people who have sustained a TBI. SEARCH STRATEGY: We searched ten electronic databases (Cochrane Injuries Group Trials Register; Cochrane Central Register of Controlled Trials (CENTRAL); EMBASE; PubMed (MEDLINE); CINAHL; AMED; SPORTDiscus; PsycINFO; PEDro and PsycBITE) and two clinical trials registers (TrialsCentral and Current Controlled Trials). The last search was August 2007. In addition we screened reference lists from included studies and contacted trialists to identify further studies. SELECTION CRITERIA: Randomised controlled studies with TBI participants were eligible if they compared an exercise programme incorporating cardiorespiratory fitness training to usual care, a non-exercise intervention or no intervention. DATA COLLECTION AND ANALYSIS: Two authors independently screened the search output, extracted data and assessed quality. All trialists were contacted for additional information. Mean difference and 95% confidence intervals (CI) were calculated for continuous data and risk difference or odds ratio and 95% CI were calculated for dichotomous data. Data were pooled when there were sufficient studies with clinical and statistical homogeneity. MAIN RESULTS: Six studies, incorporating 303 participants, were included. The participants were primarily males, in their mid thirties who had sustained a severe TBI. The studies were clinically diverse with regard to the interventions, time post-injury and the outcome measures used; therefore, the primary outcome could not be pooled. Three of the six studies indirectly assessed change in cardiorespiratory fitness after fitness training using the peak power output obtained during cycle ergometry (either at volitional fatigue or at a predetermined endpoint, that is, a percentage of predicted heart rate maximum). Cardiorespiratory fitness was improved after fitness training in one study (mean difference 59 watts, 95% CI 24 to 94), whilst there was no significant improvement in the other two studies. Four of the six studies had no drop-outs from their intervention group and no adverse events were reported in any study. AUTHORS' CONCLUSIONS: There is insufficient evidence to draw any definitive conclusions about the effects of fitness training on cardiorespiratory fitness. Whilst it appears to be a safe and accepted intervention for people with TBI, more adequately powered and well-designed studies are required to determine the effects across a range of outcome measures.

Accreditation of histocompatibility and immunogenetics laboratories: Achievements and future prospects from the European Federation for Immunogenetics Accreditation Programme.

The importance of demonstrating adherence to good practice in the provision of clinical services is well recognised, and there are many legislative and regulatory requirements that aim to ensure that services are appropriately reviewed and certified. Therefore, for regulatory purposes, laboratories must provide assurance of the quality of the services they provide. Additionally in the field of transplantation, where donor organs and stem cells are exchanged across national boundaries, adoption of a common set of standards by laboratories across many different countries is an important factor. The European Federation for Immunogenetics (EFI) Accreditation Programme was established to provide assurance that Histocompatibility & Immunogenetics laboratories providing services for transplantation, transfusion, and disease association testing meet the requirements of the specialty specific EFI standards. The first H&I laboratories achieved EFI accreditation in 1995, and currently there are over 260 EFI accredited laboratories in 36 countries. The programme depends on the voluntary participation of the inspectors, who are all experts in the field of H&I, and who, over the last 22 years, have performed over 1400 onsite inspections of laboratories. Inspection findings show the areas that are most frequently found to be deficient in meeting the requirements of the standards, and this can be used to inform educational and other activities with the aim of improving laboratory compliance with the standards. The EFI standards have been regularly updated to reflect the changes in the field with 19 versions over the last 22 years, and the data from the accreditation programme show how laboratories have changed their practices to incorporate new techniques that support patient care.

Effect of non-invasive ventilation on the measurement of ventilatory and metabolic variables.

The effect of non-invasive ventilation (NIV) on the accuracy of measurements of ventilation, oxygen consumption (V˙O2) and carbon dioxide production (V˙CO2) was examined using a simulator. Known gas volumes of oxygen and carbon dioxide were delivered to a metabolic system that measured tidal volume, respiratory rate, V˙O2 and V˙CO2, both with and without NIV. Bland-Altman analyses were used to compare between conditions. NIV at pressure support (PS) 20cm H2O compared to without NIV showed: VT, mean difference (MD) 0mL (limits of agreement (LOA) -21 to 21) mL; V˙O2 MD -413 (LOA -810 to 16) mL/min; and V˙CO2 MD 32 (LOA -32 to 97) mL/min. For V˙O2 measurements during NIV, a correction was applied to account for increased air density due to PS. After correction, V˙O2 measurement accuracy improved; MD -46 (LOA -108 to 17) mL/min. Tidal volume and metabolic variables can be measured with acceptable accuracy during NIV, providing V˙O2 is corrected for altered gas density.

Detection of HLA class I- and class II-specific antibodies by flow cytometry and PRA-STAT screening in renal transplant recipients.

BACKGROUND: Screening for HLA-specific antibodies has been performed by complement-dependent lymphocytotoxicity for many years. In recent years, methods involving the use of flow cytometry or ELISA have been developed. METHODS: This study has compared a flow cytometric screening technique for the detection of HLA class I- and class II-specific antibodies with a commercially available ELISA technique, PRA-STAT. RESULTS: A significant correlation was found between the two methods for the detection of antibodies in patients after transplantation (P<0.001). Specificity analysis confirmed that the PRA-STAT technique detected both HLA class I- and class II-specific antibodies. Screening of serum samples from patients who experienced graft loss by cytotoxic, flow cytometric, and PRA-STAT techniques showed that there was a significant correlation between all three methods for the detection of antibody, but that the best correlation for the panel-reactive antibody level was that between the flow cytometric and PRA-STAT techniques (r=0.86). This was principally due to the detection of both HLA class I- and class II-specific antibodies by these methods, whereas cytotoxic screening detected only class I-specific antibodies. CONCLUSIONS: These results suggest that PRA-STAT is a useful technique for the detection of both HLA class I- and class II-specific antibodies, rather than only class I-specific antibodies as previously described.

The clinical importance of variation within the HLA-Bw4 complex.

BACKGROUND: Antibody screening of a patient with a failed renal transplant showed positive reactions with most, but not all HLA-Bw4-associated B-locus antigens. However, the patient's serological HLA class I type suggested the presence of HLA-Bw4. METHODS: Standard molecular techniques were used to re-type the patient and donor. ELISA antibody screening helped determine the patient's antibody specificity. RESULTS: The patient's type was HLA-B*1402,4703;Bw6 and the donor HLA-B*4703,51011;Bw4,6. Analysis of ELISA results identified three amino acids (positions 77,80,81) as the most likely epitope recognised by the patient's serum. These corresponded to HLA-B*51011 amino acid mismatches, explaining the lymphocytotoxic reactivity pattern. This epitope is located on a subgroup of the HLA-Bw4 antigen suggesting anti-Bw4 was not a sufficient description of this antibody. CONCLUSIONS: This report identifies an antibody to a sub-group of the Bw4 public specificity and also confirms the need for sequence-level analysis in the tissue-typing laboratory to determine future unacceptable mismatches.

Detection of HLA-specific IGG antibodies using single recombinant HLA alleles: the MonoLISA assay.

BACKGROUND: Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS: HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS: A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION: This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.

Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study.

The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use.

Human skeletal sarcoplasmic reticulum Ca2+ uptake and muscle function with aging and strength training.

This study investigated the adaptations of skeletal muscle sarcoplasmic reticulum (SR) Ca2+ uptake, relaxation, and fiber types in young (YW) and elderly women (EW) to high-resistance training. Seventeen YW (18-32 yr) and 11 EW (64-79 yr) were assessed for 1) electrically evoked relaxation time and rate of the quadriceps femoris; and 2) maximal rates of SR Ca2+ uptake and Ca2+-ATPase activity and relative fiber-type areas, analyzed from muscle biopsies of the vastus lateralis. EW had significantly slower relaxation rates and times, decreased SR Ca2+ uptake and Ca2+-ATPase activity, and a larger relative type I fiber area than did YW. A subgroup of 9 young (YWT) and 10 elderly women (EWT) performed 12 wk of high-resistance training (8 repetition maximum) of the quadriceps and underwent identical testing procedures pre- and posttraining. EWT significantly increased their SR Ca2+ uptake and Ca2+-ATPase activity in response to training but showed no alterations in speed of relaxation or relative fiber-type areas. In YWT none of the variables was altered after resistance training. These findings suggest that 1) a reduced SR Ca2+ uptake in skeletal muscle of elderly women was partially reversed with resistance training and 2) SR Ca2+ uptake in the vastus lateralis was not the rate-limiting mechanism for the slowing of relaxation measured from electrically evoked quadriceps muscle of elderly women.

Impaired calcium pump function does not slow relaxation in human skeletal muscle after prolonged exercise.

This study examined the effects of prolonged exercise on human quadriceps muscle contractile function and homogenate sarcoplasmic reticulum Ca2+ uptake and Ca2+-adenosinetriphosphatase activity. Ten untrained men cycled at 75 +/- 2% (SE) peak oxygen consumption until exhaustion. Biopsies were taken from the right vastus lateralis muscle at rest, exhaustion, and 20 and 60 min postexercise. Peak tension and half relaxation time of the left quadriceps muscle were measured during electrically evoked twitch and tetanic contractions and a maximal voluntary isometric contraction at rest, exhaustion, and 10, 20, and 60 min postexercise. At exhaustion, homogenate Ca2+ uptake and Ca2+ adenosinetriphosphatase activity were reduced by 17 +/- 4 and 21 +/- 5%, respectively, and remained depressed after 60 min recovery (P

Skeletal muscle metabolic and ionic adaptations during intense exercise following sprint training in humans.

The effects of sprint training on muscle metabolism and ion regulation during intense exercise remain controversial. We employed a rigorous methodological approach, contrasting these responses during exercise to exhaustion and during identical work before and after training. Seven untrained men undertook 7 wk of sprint training. Subjects cycled to exhaustion at 130% pretraining peak oxygen uptake before (PreExh) and after training (PostExh), as well as performing another posttraining test identical to PreExh (PostMatch). Biopsies were taken at rest and immediately postexercise. After training in PostMatch, muscle and plasma lactate (Lac(-)) and H(+) concentrations, anaerobic ATP production rate, glycogen and ATP degradation, IMP accumulation, and peak plasma K(+) and norepinephrine concentrations were reduced (P<0.05). In PostExh, time to exhaustion was 21% greater than PreExh (P<0.001); however, muscle Lac(-) accumulation was unchanged; muscle H(+) concentration, ATP degradation, IMP accumulation, and anaerobic ATP production rate were reduced; and plasma Lac(-), norepinephrine, and H(+) concentrations were higher (P<0.05). Sprint training resulted in reduced anaerobic ATP generation during intense exercise, suggesting that aerobic metabolism was enhanced, which may allow increased time to fatigue.

Influence of negative childhood experiences on psychological functioning, social support, and parenting for mothers recovering from addiction.

OBJECTIVE: This study was conducted with mothers recovering from drug and alcohol addiction and had three aims: first, to understand the range of negative childhood events these mothers experienced; second, to understand their current level of distress and their parenting experiences; and third, to examine the relationships between negative childhood events and parenting experiences. METHOD: Forty-six mothers participated in a cross-sectional exploratory study and completed a range of self-report measures, including the Child Abuse & Trauma Scale, Social Support Inventory, CES-D, Parenting Stress Index, and the Parenting Scale. RESULTS: When compared to norming samples these mothers reported significantly higher levels of aversive childhood experiences, psychological distress, parenting stress and use of problematic parenting behaviors along with lower levels of social support. Higher levels of neglect and growing up in a negative home environment were significantly correlated with lower levels of social support from the family, higher levels of distress and parenting stress, and greater use of problematic parenting behaviors. CONCLUSION: For this sample there is a greater incidence of aversive childhood experiences and greater problems with maternal functioning. Mothers recovering from addiction have an additional need for clinical attention towards issues of recovery from childhood abuse and responding to parenting difficulties with their own children.

Renal disease, epidermal necrosis, and epithelial cell antibodies.

OBJECTIVE: To describe the association between epithelial cell IgM, which has previously been associated with an increased incidence of loss of renal graft in children, with a novel cutaneous eruption and unexplained native renal disease. DESIGN: Observational study on children with epithelial cell antibody presenting with unexplained renal or skin disease. SETTING: General paediatric department and regional paediatric nephrology unit. PATIENTS: Six children (five girls, one boy), who presented to the unit in 1989-90. RESULTS: Three children, two of whom had a history of a hyperpigmented rash, presented with hypertension, proteinuria, and impaired renal function. Renal biopsy specimens from two of these children showed severe arteriolar endothelial cell swelling with arteriolar occlusion. These children fully recovered after treatment with antihypertensive drugs. The third child developed end stage renal failure and required dialysis. Three other children presented with an unusual cutaneous eruption but no evidence of renal disease. Histology of the skin lesions showed acute epidermal necrosis and features consistent with a viral infection. CONCLUSIONS: The aetiology and pathogenesis of the epithelial cell antibody are unknown. These cases indicate that it may have a role in native kidney disease and focal epidermal necrosis. Clinical and histological features suggest that the antibody may be associated with a viral infection.

The contribution of VEGF signalling to fostamatinib-induced blood pressure elevation.

BACKGROUND AND PURPOSE: Fostamatinib is an inhibitor of spleen tyrosine kinase (TK). In patients, fostamatinib treatment was associated with increased BP. Some TK inhibitors cause BP elevation, by inhibiting the VEGF receptor 2 (VEGFR2). Here, we have assessed the mechanistic link between fostamatinib-induced BP elevation and inhibition of VEGF signalling. EXPERIMENTAL APPROACH: We used conscious rats with automated blood sampling and radio telemetry and anaesthetized rats to measure cardiovascular changes. Rat isolated aorta and isolated hearts, and human resistance vessels in vitro were also used. NO production by human microvascular endothelial cells was measured with the NO-dependent probe, DAF-FM and VEGFR2 phosphorylation was determined in mouse lung, ex vivo. KEY RESULTS: In conscious rats, fostamatinib dose-dependently increased BP. The time course of the BP effect correlated closely with the plasma concentrations of R406 (the active metabolite of fostamatinib). In anaesthetized rats, infusion of R406 increased BP and decreased femoral arterial conductance. Endothelial function was unaffected, as infusion of R406 did not inhibit hyperaemia- or ACh-induced vasodilatation in rats. R406 did not affect contraction of isolated blood vessels. R406 inhibited VEGF-stimulated NO production from human endothelial cells in vitro, and treatment with R406 inhibited VEGFR2 phosphorylation in vivo. R406 inhibited VEGF-induced hypotension in anaesthetized rats. CONCLUSIONS AND IMPLICATIONS: Increased vascular resistance, secondary to reduced VEGF-induced NO release from endothelium, may contribute to BP increases observed with fostamatanib. This is consistent with the elevated BP induced by other drugs inhibiting VEGF signalling, although the contribution of other mechanisms cannot be excluded.