Characterising and justifying sample size sufficiency in interview-based studies: systematic analysis of qualitative health research over a 15-year period.

BACKGROUND: Choosing a suitable sample size in qualitative research is an area of conceptual debate and practical uncertainty. That sample size principles, guidelines and tools have been developed to enable researchers to set, and justify the acceptability of, their sample size is an indication that the issue constitutes an important marker of the quality of qualitative research. Nevertheless, research shows that sample size sufficiency reporting is often poor, if not absent, across a range of disciplinary fields. METHODS: A systematic analysis of single-interview-per-participant designs within three health-related journals from the disciplines of psychology, sociology and medicine, over a 15-year period, was conducted to examine whether and how sample sizes were justified and how sample size was characterised and discussed by authors. Data pertinent to sample size were extracted and analysed using qualitative and quantitative analytic techniques. RESULTS: Our findings demonstrate that provision of sample size justifications in qualitative health research is limited; is not contingent on the number of interviews; and relates to the journal of publication. Defence of sample size was most frequently supported across all three journals with reference to the principle of saturation and to pragmatic considerations. Qualitative sample sizes were predominantly - and often without justification - characterised as insufficient (i.e., 'small') and discussed in the context of study limitations. Sample size insufficiency was seen to threaten the validity and generalizability of studies' results, with the latter being frequently conceived in nomothetic terms. CONCLUSIONS: We recommend, firstly, that qualitative health researchers be more transparent about evaluations of their sample size sufficiency, situating these within broader and more encompassing assessments of data adequacy. Secondly, we invite researchers critically to consider how saturation parameters found in prior methodological studies and sample size community norms might best inform, and apply to, their own project and encourage that data adequacy is best appraised with reference to features that are intrinsic to the study at hand. Finally, those reviewing papers have a vital role in supporting and encouraging transparent study-specific reporting.

An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics.

There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.

An International Standard for holotranscobalamin (holoTC): international collaborative study to assign a holoTC value to the International Standard for vitamin B12 and serum folate.

BACKGROUND: Investigation of possible B12 and folate deficiencies requires measurement of these vitamins in serum. There is evidence that holotranscobalamin (holoTC), the active portion of B12 available to cells, is a more specific marker of early B12 deficiency than total B12. The availability of immunoassays for holoTC prompted an international collaborative study to assign a holoTC value to the World Health Organization (WHO) 1st International Standard (IS) for vitamin B12 and serum folate, 03/178. METHODS: The IS, 03/178, and three serum samples with different holoTC levels were assayed by 12 laboratories in eight countries using manual and automated immunoassays for holoTC; one laboratory additionally performed an in-house assay. Fourteen sets of data were analysed. RESULTS: Overall, the IS, 03/178, and the three serum samples demonstrated assay linearity and parallelism. An overall geometric mean (GM) holoTC value of 106.8 pmol/L was obtained for 03/178, with an inter-laboratory geometric coefficient of variation (GCV) of 10.5%. There was a reduction in inter-laboratory variability when the holoTC levels in the serum samples were determined relative to the IS with an assigned holoTC value rather than to the assays' calibration. Accelerated degradation studies showed that 03/178 was sufficiently stable to serve as an IS for holoTC. CONCLUSIONS: The WHO Expert Committee on Biological Standardization endorsed the proposal to assign a holoTC value of 107 pmol/L to 03/178, corresponding to 0.107 pmol per ampoule, for use as the 1st IS for vitamin B12, serum folate, and holoTC.

Specifications for anti-A and anti-B in intravenous immunoglobulin: history and rationale.

Intravenous immunoglobulin (IVIG) products are generally safe and efficacious, although treatment-related adverse reactions can occur in recipients. Adverse reactions include hemolysis in non-blood group O recipients linked to the passive transfer of anti-A and/or anti-B present in the fractionated immunoglobulin product. In light of the recent increase in reported cases of severe hemolysis associated with anti-A and/or anti-B, this article traces the development of pharmacopoeial specifications, tests, and reference reagents to control their titers in IVIG products.

The 3rd International Standard for serum IgE: international collaborative study to evaluate a candidate preparation.

BACKGROUND: The measurement of serum IgE aids in the diagnosis and management of atopic allergic disease and hyper-IgE immunodeficiency syndromes. The 2nd World Health Organization (WHO) International Reference Reagent (IRR) for serum IgE (75/502; 5000 IU/ampoule), is widely used to calibrate assays for serum IgE. Exhaustion of stocks of the 2nd IRR necessitated the production of a replacement preparation and its evaluation in an international collaborative study to determine its suitability to serve as the 3rd International Standard (IS) for serum IgE. METHODS: Sera and defibrinated plasma with elevated IgE levels were pooled and lyophilised in ampoules. This preparation, coded 11/234, was assayed by 18 laboratories in 11 countries using commercial assay methodology for IgE, along with the 2nd IRR, 75/502, and two lyophilised serum samples. RESULTS: Overall, there were no consistent differences in the way that the candidate IS (11/234), the IRR (75/502), and the two serum samples behaved in the assays with respect to linearity and parallelism. The mean IgE value of the candidate IS, 11/234, relative to the IRR, 75/502, was 13,411 IU/mL based on parallel line analysis of raw assay data at NIBSC, and 13,551 IU/mL based on the laboratories' own estimates after correcting for the values obtained for 75/502. CONCLUSIONS: The use of 11/234 will ensure that assays for serum IgE continue to be well standardised. The preparation was established by the WHO Expert Committee on Biological Standardization as the 3rd IS for serum IgE with an assigned value of 13,500 IU/mL, corresponding to 6750 IU/ampoule.

Antibody C region influences TGN1412-like functional activity in vitro.

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.

Priming moral self-ambivalence heightens deliberative behaviour in self-ambivalent individuals.

BACKGROUND: Recent work on cognitive-behavioural models of obsessive-compulsive disorder has focused on the roles played by various aspects of self-perception. In particular, moral self-ambivalence has been found to be associated with obsessive-compulsive phenomena. AIMS: In this study we used an experimental task to investigate whether artificially priming moral self-ambivalence would increase participants' deliberation on ethical problems, an index that might be analogous to obsessive-compulsive behaviour. METHOD: Non-clinical participants completed two online tasks designed to prime either moral self-ambivalence, general uncertainty, or neither. All participants then completed a task requiring them to consider solutions to moral dilemmas. We recorded the time participants took to respond to the dilemmas and the length of their responses; we then combined these variables to create a measure of deliberation. RESULTS: Priming moral self-ambivalence led to increases in deliberation, but this was only significant among those participants who scored highly on a baseline measure of moral self-ambivalence. Priming general uncertainty had no significant effect upon deliberation. CONCLUSIONS: The results suggest that moral self-ambivalence may play a role in the maintenance of obsessive-compulsive behaviour. We propose that individuals who are morally self-ambivalent might respond to situations in which this ambivalence is made salient by exhibiting behaviour with obsessive-compulsive characteristics. These findings have implications for the incorporation of ideas about self-concept into theories of obsessive-compulsive disorder.

Severity of the TGN1412 trial disaster cytokine storm correlated with IL-2 release.

AIM: To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. METHODS: Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. RESULTS: Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894-6051 pg ml⁻¹) compared with muromonab-CD3 (62-262 pg ml⁻¹) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. CONCLUSIONS: The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.

Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture.

The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.

Consistent manufacturing and quality control of a highly complex recombinant polyclonal antibody product for human therapeutic use.

The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.

The mediating roles of disgust sensitivity and danger expectancy in relation to hand washing behaviour.

BACKGROUND: Recent interest in the role of vulnerability factors in obsessional washing has suggested that disgust sensitivity, danger expectancy and health anxiety may be of interest. AIMS: This study explores the differential impact of these factors on both behavioural and cognitive measures of washing behaviour and is based on a replication of the Jones and Menzies (1997) experiment, during which participants immersed their hands in a noxious compound while rating themselves on a range of measures: the time they subsequently took to wash their hands was measured and danger expectancies were found to be the best predictor of this. METHOD: The present study added measures of disgust sensitivity and health anxiety to this experimental methodology while removing factors they found to be of little import to compulsive washing. Thirty non-clinical participants took part. RESULTS: Results confirmed that disgust sensitivity was related to the behavioural measure of washing time, but that this relationship was almost entirely mediated by the danger expectancy concerning judgements of severity of consequent disease. However, a different pattern emerged when the outcome measure was questionnaire based: danger expectancy was not at all related to this. Disgust sensitivity mediated the relationship between health anxiety and scores on a questionnaire measure of washing compulsions. Interestingly, these scores were not related to the behavioural measure of washing time. CONCLUSIONS: The implications of these relationships to the further development of subtypes of Obsessive Compulsive Disorder (OCD) are discussed.

A WHO reference reagent for the Serum Transferrin Receptor (sTfR): international collaborative study to evaluate a recombinant soluble transferrin receptor preparation.

BACKGROUND: The usefulness of serum transferrin receptor (sTfR) as a marker of iron deficiency is limited by lack of standardization of commercial immunoassays for sTfR. An international collaborative study was performed to evaluate a lyophilized preparation of recombinant soluble transferrin receptor (rsTfR) for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize immunoassays for sTfR. METHODS: The concentration of pure rsTfR was determined from the A(280 nm) using the adjusted theoretical extinction coefficient and molecular weight calculated from its sequence, before dilution and lyophilization in a sTfR-depleted serum matrix. Six manufacturers and a health protection laboratory assayed the candidate Reference Reagent, coded 07/202, along with three lyophilized serum samples, using commercial assays for sTfR. RESULTS: Dose-response plots demonstrated acceptable overall parallelism between 07/202, manufacturers' in-house standards, and serum samples. However, there was poor agreement on the estimated (r)sTfR content of 07/202 and serum samples. Expressing the sTfR content of the serum samples relative to 07/202 markedly improved agreement between methods. CONCLUSIONS: Use of 07/202 would reduce inter-method variability. The preparation was established as the 1st WHO Reference Reagent for sTfR with assigned free rsTfR monomer values of 21.7 mg/L and 303 nmol/L (0.5 mL reconstitution).

The development and role of international biological reference materials in the diagnosis of anaemia.

Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B12), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B12 and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.

Exploring aspects of the cognitive behavioural model of physical hoarding in relation to digital hoarding behaviours.

While the hoarding of physical objects has been extensively explored, there is little research relating to the hoarding of digital materials. The research that has been conducted suggests that digital hoarding (DH) behaviours appear to have some similarities with physical hoarding (PH) behaviours, and can be just as psychologically distressing. This study uses the framework of the cognitive behavioural model of PH to explore DH behaviours, including possible similarities regarding emotional attachment to digital possessions, and possible links with Obsessive Compulsive Disorder (OCD) and indecisiveness. For the study, 282 participants completed an online survey which measured levels of digital and physical hoarding, compulsive acquisition, OCD, indecisiveness and mood. Strong emotional attachments to particular types of digital possessions were evident: this was especially true for photographs and videos. Significant positive relationships were found between all the variables measured. However, a regression analysis revealed that only OCD and PH scores were significant predictors of DH. DH thus appears to share some of the features of PH. Implications, limitations and future research possibilities are discussed.

Claustrophobia in MRI: the role of cognitions.

PURPOSE: This study aimed to investigate the role of cognitive and behavioural factors in the experience of claustrophobia in the context of magnetic resonance imaging (MRI) scanners. MATERIALS AND METHODS: One hundred and thirty outpatients attending an MRI unit completed questionnaires before and after their scans. Specific measures of experience in the scanner included subjective anxiety, panic symptoms, strategies used to stay calm and negative cognitions (such as 'I will suffocate' and 'I am going to faint in here'). Other general measures used included anxiety, depression, health anxiety and fears of restriction and suffocation. RESULTS: The amount of anxiety experienced during the scan was related to the perceived amount of time spent having physical symptoms of panic. Cognitions reported concerned the following: suffocation, harm caused by the machine and lack of perceived control. The number of strategies patients used to cope in the machine was also a related factor. Neither position in the scanner, nor head coil use nor previous experience of being in the scanner was related to levels of anxiety. CONCLUSION: The cognitions identified here may be used to construct a measure to identify those unable to enter the scanner or those most likely to become claustrophobic whilst undergoing the procedure and to further inform future brief, effective interventions.

"Perhaps you only imagined doing it": reality-monitoring in obsessive-compulsive checkers using semi-idiographic stimuli.

Memory failures reported by obsessive-compulsive (OC) checkers often seem to be errors of "reality-monitoring", or misremembering whether one performed or imagined performing an action. To examine these memory processes in the context in which such errors are said to occur, an in-home reality-monitoring experiment involving bothersome and non-bothersome actions was conducted with 21 OC checkers and 24 non-clinical controls. OC checkers reported poorer confidence in memory, but both groups performed similarly on tests of immediate and delayed free and prompted recall. Among OC checkers (but not controls), accuracy in recall and confidence in memory were correlated. Theoretical implications are discussed.

A VH4-34+ myeloma protein with weak autoreactivity.

It has been suggested that VH4-34 gene segment expression is counter-selected in multiple myeloma (MM) due to a self-tolerance mechanism. We cloned and sequenced a VH4-34 gene segment from bone marrow mononuclear cells of a stage III MM patient. We show that VH4-34 was expressed by the serum IgA myeloma (M)-protein, as demonstrated by reactivity with the VH4-34 specific 9G4 mAb and mass spectrometry (MS). The M-protein had weak reactivity with nuclei. These results demonstrate that VH4-34 may be expressed in secreted IgA M-protein with weak autoreactivity. Thus, counter-selection of VH4-34 is pronounced but not absolute in MM. Mechanisms of how VH4-34 can occasionally be expressed in MM and clinical implications are discussed.

Advanced glycation end product interventions reduce diabetes-accelerated atherosclerosis.

Advanced glycation end product (AGE) formation may contribute to the progression of atherosclerosis, particularly in diabetes. The present study explored atherosclerosis in streptozotocin-induced diabetic apolipoprotein E-deficient (apoE-/-) mice that were randomized (n = 20) to receive for 20 weeks no treatment, the AGE cross-link breaker ALT-711, or the inhibitor of AGE formation aminoguanidine (AG). A sixfold increase in plaque area with diabetes was attenuated by 30% with ALT-711 and by 40% in AG-treated mice. Regional distribution of plaque demonstrated no reduction in plaque area or complexity within the aortic arch with treatment, in contrast to the thoracic and abdominal aortas, where significant attenuation was seen. Diabetes-associated accumulation of AGEs in aortas and plasma and decreases in skin collagen solubility were ameliorated by both treatments, in addition to reductions in the vascular receptor for AGE. Collagen-associated reductions in the AGEs carboxymethyllysine and carboxyethyllysine were identified with both treatments. Diabetes was also accompanied by aortic accumulation of total collagen, specifically collagens I, III, and IV, as well as increases in the profibrotic cytokines transforming growth factor-beta and connective tissue growth factor and in cellular alpha-smooth muscle actin. Attenuation of these changes was seen in both treated diabetic groups. ALT-711 and AG demonstrated the ability to reduce vascular AGE accumulation in addition to attenuating atherosclerosis in these diabetic mice.

Selective cleavage of human IgG by the matrix metalloproteinases, matrilysin and stromelysin.

We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH(2) domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fabprime prime or minute)(2) from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflamed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.