Validation of high-performance liquid chromatography-tandem mass spectrometry assays quantifying omacetaxine mepesuccinate and its 4'­des-methyl and cephalotaxine metabolites in human plasma and urine.

Omacetaxine mepesuccinate (hereafter called omacetaxine) is a modified cephalotaxine and is registered (Synribo(®)) for the treatment of adult patients with chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs). To evaluate the pharmacokinetics of omacetaxine, sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for the quantification of omacetaxine and its inactive 4'-des-methyl (4'-DMHHT) and cephalotaxine metabolites in human plasma and urine were developed and validated. Since omacetaxine is mainly metabolised by esterases, the plasma samples were immediately stabilised after collection with an esterase inhibitor and stored at a nominal temperature of -80°C. Urine samples were stored at -80°C immediately after collection. Protein precipitation was applied as the sample pretreatment method for the plasma samples, and urine samples were processed using solid-phase extraction (SPE). For both assays, the dried and reconstituted extracts were injected on a XBridge BEH Phenyl column for analysis of all analytes. Gradient elution was applied with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionised using a turbospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The validated plasma assay quantifies all analytes in the concentration range of 0.1-100ng/mL and the urine assay in the range of 0.1-50ng/mL. At all concentrations, the accuracies were within ±15% of the nominal concentrations and precisions were ≤15%. The developed methods have successfully been applied in a human mass balance study of omacetaxine.

Practice learning teams: a partnership approach to supporting students' clinical learning.

The importance of clinical learning for students has been acknowledged by both government and nursing regulatory bodies who have called for partnerships and collaborative structures to be developed to facilitate the provision of good quality structured support for all learners in practice placements [Department of Health DOH, Making a Difference: Strengthening the Nursing, Midwifery and Health Visiting Contribution to Health and Health Care, Department of Health, London, 1999; ; United Kingdom Central Council for Nursing, Midwifery and Health Visiting; UKCC, Fitness for Practice: The UKCC Commission for Nursing and Midwifery Education, 1999; United Kingdom Central Council for Nursing, Midwifery and Health Visiting. London; ; ENB/DoH, Placements in Focus. English National Board for Nursing, Midwifery and Health Visiting, London, (2001b)]. This paper reports the early experiences of developing and implementing one such collaborative approach in one School of Nursing in England. The approach presented is that of practice learning teams (PLTs). The driving forces behind the decision to implement these teams and why the change was thought to be necessary when another approach, namely the link lecturer role was already in existence are considered. The challenges encountered during the implementation process and the perceived benefits that are emerging are discussed.

A 384-well solid-phase extraction for LC/MS/MS determination of methotrexate and its 7-hydroxy metabolite in human urine and plasma.

A solid-phase extraction procedure, in a 384-well format, has been developed for methotrexate and its primary metabolite, 7-hydroxymethotrexate, in human urine and plasma. This format has not been utilized previously for solid-phase extraction of drugs from biological fluids. The 384-well plates contained a C-18 stationary phase bonded to silica particles which are incorporated into a glass-fiber membrane. Methotrexate and 7-hydroxymethotrexate have been quantified across the curve range of 1 to 50 microg/mL and 50 to 1000 ng/mL, respectively, in urine and from 5 to 250 ng/mL and 5 to 100 ng/mL, respectively, in plasma. Both analytes are quantified by linear regression using 20-microL sample aliquots. Experiments to evaluate the influence of particle size, elution volume, and injection volume on signal intensity were conducted and are reported, along with the results of experiments examining cross contamination between wells. Recovery was determined to be > or = 95% from urine. Results from a run of 384 samples analyzed over a 14-h period indicate that 384-well SPE can be successfully utilized to increase analytical run sizes and sample throughput for LC/MS/MS determination of small drug molecules in biological samples.

Secrecy, disclosure, and closet dynamics.

This paper examines the assumption that male homosexuality has a natural affinity with femininity and that male heterosexuality has a natural affinity with masculinity. An analysis of the relationship between people's disclosure or concealment of their homosexual practice or identity, particularly as it relates to notions of hegemonic masculinity and femininity provides the focus of this paper. It is argued that everyday understandings of homosexuality tend to be resolved in such as way as to press homosexuality into the service of privileging a male, masculine, and heterosexual subjectivity. This privileging is achieved, in part, as a result of the everyday social practices of homosexually active men's witting and unwitting deference to the hegemonic presumption that masculine men are naturally heterosexual, and its inverse, that feminine men are homosexual and are a perturbation of the natural order. We argue that this correlation is manufactured in everyday life in the world of appearances, but that the appearance of things is not reflected at a level of practice, which is to say, male homosexual practice is not necessarily feminine, just as male heterosexual practice is not necessarily masculine. Realities that conflict with the hegemonic realities are masked in the public world, for a variety of reasons. What we have called closet dynamics are the various discourses through which homosexuality is concealed and disclosed, and the various subject positions people take up in relation to those discourses.

"Semi-straight sort of sex": class and gay community attachment explored within a framework of older homosexually active men.

Gay Community Attachment has proved a significant predictor of successful behavior change among gay-identifying men in response to HIV/AIDS. Related work at Macquarie University, Sydney, Australia, indicated that attachment to gay community is not a simple issue; rather, complex issues of sexual identity formation, the constraints of social inequality and localized sexual cultures inhibit the process of attachment and, therefore, successful HIV prevention. This paper discusses some of the findings from close-focus (qualitative) research on older homosexually active men which explore in depth the dynamic whereby these men attached themselves to gay community in terms of an analysis of class, generation, and the interplay with self-construction and masculinity.

A survey of the teaching and learning of biological sciences on undergraduate nursing courses.

Curriculum planners developing degree courses in nursing have to decide how much time to allocate to each of the academic disciplines including biological sciences. There is no research-based evidence to suggest what depth and detail of knowledge of biological sciences is required to support nursing practice. There is also some debate about the teaching methods used and who should teach the biological sciences. This paper reports the results of a small survey investigating the teaching of biological sciences on 16 nursing degree courses in the UK. The survey uncovered great variation in the number of hours spent on biological sciences in the different universities and in the science entry requirements of the different universities. Most teachers of biological sciences had a first degree in the subject but few were nurses. The possible implications of these findings are discussed. Problems associated with shared learning and didactic teaching methods are also highlighted. Although the biological sciences input will largely be a matter of institutional preferences, nursing needs to develop a research-based framework to aid curriculum planning.

Bachelor of nursing students' experiences of learning biological sciences alongside medical students.

One of the features of the Bachelor of Nursing (BN) course at Nottingham University is that the students study some biological sciences alongside their medical counterparts. Formative evaluation of the processes of teaching and learning, with a view to monitoring and improving the process of education taking place, is an integral part of the BN curriculum. The findings obtained to date suggest that, whilst the students value some aspects of learning biological sciences with medical students, they are anxious about the effectiveness of the teaching methods being employed; the difficulty of what they are learning and the depth and level of knowledge required in order to deliver safe competent nursing care. They are also concerned about the medical bias of the content that they are being taught and its relevance for them as nurses. This paper will set the biological sciences component of the course in context within both the BN and the medical curriculum. The methods being used to evaluate the teaching and learning processes and the results of these evaluations will be discussed and the implications of the students' experiences will be explored.

Avascular necrosis after treatment of aplastic anaemia with antilymphocyte globulin and high-dose methylprednisolone.

Avascular necrosis of bone (AVN) occurring in patients with aplastic anaemia (AA) treated with antilymphocyte globulin (ALG) followed by high-dose methylprednisolone (HDMP) has been studied retrospectively. Out of 49 patients treated at two centres, seven have developed AVN at a median of 14 months (range 6-30) following treatment. The cumulative incidence of AVN is 21% (95% confidence intervals 7-35%). The hip was involved in six patients, bilaterally in five. Two patients had more than two joints affected. Surgical intervention was necessary in five patients, three of whom required total hip replacement. In contrast, there were no cases of AVN in a historical group of 61 patients with aplastic anaemia treated with an identical regimen of ALG but using a short course of low-dose prednisolone. Lack of convincing evidence for benefit, the considerable morbidity from AVN, and increased risk of early life-threatening infection, suggest that high-dose methyl prednisolone should be omitted from ALG treatment protocols for aplastic anaemia.

Fluorescence quenching; a practical problem in flow cytometry.

Dual fluorescence analysis with a single-laser fluorescence-activated cell sorter is dependent on the use of two fluorochromes with similar excitation wavelengths but different emission wavelengths. The dye pair fluorescein and R-phycoerythrin (RPE) have been widely employed for this purpose and interaction between the two dyes has not been observed. Here evidence is presented to show that at high concentrations RPE can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction in the fluorescein signal correlated with the intensity of red (RPE) staining. This phenomenon can seriously compromise interpretation of dual immunofluorescence carried out on a single laser instrument and can best be avoided by careful analysis of single colour controls.

A phenotypic study of B lymphocyte subpopulations in human bone marrow.

The regulatory mechanisms that monitor the size of the peripheral B cell pool and determine cell death or survival are poorly understood. In rodents B lymphopoiesis is maintained at a high rate throughout adult life, and under resting conditions there is little recruitment into the long-lived peripheral pool; it therefore follows that most newly formed B lymphocytes have a very short lifespan. The maturation stages of B lymphopoiesis in humans and in experimental mammals appear to be similar. We have determined the phenotype of sIgM- and sIgD-expressing cells from normal adult human bone-marrow and peripheral blood by dual immunofluorescence with an extensive panel of monoclonal antibodies representative of major B cell clusters, in order to identify antigenic differences that may play a regulatory role. Antibodies of the CD21, CD22 and CD9 clusters, the unclustered restricted B antibody 7-F-7 and anti-IgD were reactive with different proportions of sIgM+ cells in blood and bone marrow; 29.5% (range 5-60%) of sIgM+ cells in marrow were sIgD- and most of these cells were also CD21- and CD22-, thus defining a unique marrow population. However, newly formed and mature re-circulating cells comprising the sIgM+sIgD+ population could not be distinguished by the panel of antibodies.

Bone marrow transplantation for Fanconi's anaemia: the Hammersmith experience 1977-89.

Twenty-one patients with Fanconi's anaemia (FA) were treated by allogeneic bone marrow transplantation (BMT). Two, transplanted before 1980, received high dose cyclophosphamide conditioning and both died. Subsequently 19 patients received conditioning with low dose cyclophosphamide 5 mg/kg x 4 and total body irradiation 200 cGy x 3. Ten of 19 received HLA identical sibling marrow (ID-BMT) and nine marrow from alternative donors (MM-BMT). Marrow was T cell depleted in 9/19 cases. Sustained engraftment was observed in 13 cases (eight ID-BMT, five MM-BMT). Nine patients developed greater than or equal to grade II acute graft-versus-host disease (GVHD) (six ID-BMT, nine MM-BMT). Chronic GVHD occurred in 5/11 evaluable patients. Overall survival of the low dose cyclophosphamide group was 9/19 (47%) at a median follow-up of 1257 days post-BMT (110-1825). Six of 10 (60%) survived after ID-BMT compared with two of nine (22%) after MM-BMT. We conclude that allogeneic BMT using a low dose cyclophosphamide protocol is a satisfactory treatment for FA patients who have a normal HLA identical sibling. The results of MM-BMT have been poor, and must improve before these transplants can be generally recommended for treatment of FA.

Fluorescence quenching of fluorescein by R-phycoerythrin. A pitfall in dual fluorescence analysis.

The use of a single laser fluorescence-activated cell sorter (FACS) to analyse cellular subpopulations by immunochemical staining requires an alternative dye to fluorescein with appropriate spectral characteristics. R-phycoerythrin (RPE) is widely employed for this purpose. In this study the ability of RPE to quench the fluorescein emission when both are attached to the same cell has been demonstrated by dual labelling of tonsil lymphocytes with pairs of monoclonal antibodies. Reduction of the fluorescein signal correlated with the amount of RPE attached and the relative intensity of emission from the two fluorochromes. The possible photochemical mechanisms which result in a reduction of the fluorescein signal by RPE are discussed. The inclusion of control tests, in which RPE is omitted, is recommended in order to avoid misinterpretation of the results of subpopulation analysis by single laser FACS - especially when low levels of fluorescein staining are obtained.

Joint contractures and diabetic retinopathy.

It has been suggested that joint contractures may be an early marker of microangiopathy, especially retinopathy, in diabetic patients. To investigate this possibility, the prevalence of contractures of the finger joints, as assessed by the painted hand technique, was compared in 106 diabetic patients without, and 105 with retinopathy (proliferative in 66%). There was an increased prevalence of contractures in those with diabetes (25.2%) as compared with an age-matched control of 106 subjects (7.5%, P less than 0.01). The prevalence of contractures was similar in those diabetics with and without retinopathy (29.5% v. 20.8%, P less than 0.1), and did not vary with the type of retinopathy. The contracture prevalence rate was also similar in those with a diabetes duration of 5 or more years (retinopathy 29.3%, no retinopathy, 30%) or of similar diabetic duration (retinopathy 20.8%, no retinopathy, 25%). Diabetic control, as assessed by HbA1, was similar in retinopathic patients with or without contractures. We conclude that although finger joint contractures are more prevalent in adult diabetic patients, they are not necessarily a reliable early indicator of the development of retinopathy of any specific type.

The international postpartum family planning program: eight years of experience.

The International Postpartum Program was begun in 1966 in order to demonstrate the feasibility of providing efficient and effective family planning services in the context of the obstetrical care provided by hospitals. The project included 138 institutions in 21 countries. Over an eight-year period, 1.14 million women were recruited, representing 33 acceptors per 100 obstetrical/abortion patients in these hospitals. Relying primarily on modern methods of contraception, the pill, the IUD, and sterilization, the program provided about 124 years of contraceptive protection for each 100 obstetrical/abortion patients, or somewhat more than four years' protection per acceptor. Predischarge insertion of IUDs was found to be safe and effective. In all countries, the program acceptors experienced important changes in fertility over and above the effects of aging. The demonstration showed that basing a program on activities in the obstetrical wards permits a rapid buildup of services, without great cost for construction. Whether using specially trained additional staff to provide family planning education and information or relying on the existing staff, hospitals were able to recruit a substantial proportion of women on the maternity wards as acceptors. Cost per acceptor averaged about US$5.00 during the eight-year period--considerably below the cost per acceptor in the majority of national programs. Such economy arose because acceptance ratios for the International Postpartum Program were higher than ratios for national programs where the target population is more dispersed, and because only direct costs for the services needed to be considered. The idea of uniting family planning information and services with the maternity services of hospitals has been seized upon and replicated outside the International Postpartum Program. The governments of India, Pakistan, Indonesia, Colombia, Thailand, and the Philippines, among others, now operate such systems. Postpartum programs still do not reach substantial segments of the urban population in the developing world, but the expansion of such services is continuing. Also, adaptations of maternity-centered family planning programs are now being tested in rural areas in the world, where most children are born.

Gall-bladder emptying measured by a radioisotopic method.

A method of oral radioisotope cholecystography is described using 131I sodium ipodate. Gall-bladder emptying was measured by this method and radiographically: (a) in a model, (b) in 34 patients having oral cholecystography. There was a good correlation between the two methods of measuring volume changes, both in vitro and in vivo. The isotope cholecystogram has the advantage of a lower radiation hazard, and is the only satisfactory method of studying the onset, rate and duration of gall-bladder emptying. These may be easily measurable markers of disorders of the upper small bowel.