Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction.

Allogeneic bone marrow transplantation (BMT) with marrow ablative conditioning is the treatment of choice for haematopoietic malignancies. The use of nonmyeloablative stem cell transplants has allowed the treatment of patients previously ineligible for BMT because of age or other disease. These reduced conditioning regimes allow the persistence initially of some recipient cells in the blood and bone marrow (haematopoietic chimaerism). Monitoring of the relative proportion of donor and recipient cells is required to assess the success of the procedure, to predict subsequent rejection or impending relapse and to guide the use of donor lymphocyte infusions. We present a quantitative real-time PCR approach for the measurement of haematopoietic chimaerism using the TaqMan. This approach exploits the presence of single-nucleotide polymorphisms (SNPs) to distinguish cells of patient or donor origin. We have designed and validated a panel of seven allele-specific probes to quantify the contribution of patient and donor cells in the haematopoietic population from 12 patient and donor pairs. We have compared the performance of this approach with an existing method and proved it to be superior in both accuracy and sensitivity. The use of more sensitive and accurate techniques permits earlier intervention for improved clinical outcome.

Appendicular skeletal status and hip fracture in the elderly: 14-year prospective data.

Fourteen-year follow-up of 535 elderly people included in a British survey in 1973 revealed 23 incident hip fractures. The relationship between appendicular bone mass, assessed in the initial survey by metacarpal morphometry, and fracture risk was analyzed. There was an increased risk of hip fracture with declining metacarpal cortical index at baseline. The risk increase, however, was not statistically significant. It remained similar after adjustment was made for the reported prevalence of falls. These prospective data suggest that osteoporosis may contribute less to the risk of hip fracture in the very elderly than in younger individuals.

Hip fracture in Hong Kong and Britain.

Hip fractures in elderly people are an important public health problem in many Western populations. Little is known of their epidemiology in Oriental populations. A comparative study of hip fracture incidence in Hong Kong and Southampton showed similar incidence rates by age and sex. In the last two decades hip fracture incidence in Hong Kong has increased, particularly among men. This can be partly explained by reduction in the levels of activity among the elderly Chinese population.

Water fluoride concentration and fracture of the proximal femur.

STUDY OBJECTIVE: The aim of the study was to examine the relationship between water fluoride concentration and the incidence of hip fracture, since evidence on this is at present inconsistent. DESIGN: Numbers of hospital admissions for fractures of proximal femur were obtained from hospital activity analysis data for the years 1978-1982. The fracture rates were compared with water fluoride concentrations in 39 county districts of England and Wales (fluoride concentrations had been measured in these districts between 1969 and 1973 as part of the British Regional Heart Study). PATIENTS: During the study period, 4121 men and 16,272 women aged 45 years and over were discharged from hospital after hip fracture. RESULTS: Poor correlations were found between discharge rates and both total (r = 0.16, p = 0.34) and natural (r = 0.01, p = 0.95) water fluoride concentrations. CONCLUSIONS: Water fluoridation to levels of around 1 mg/litre is unlikely to reduce hip fracture incidence markedly in this country.

Quantitation of cyclin D1 over-expression using competitive fluorescent reverse transcription polymerase chain reaction: a tool for the differential diagnosis of mantle cell lymphoma.

Mantle cell lymphoma is characterized by the presence of the t(11;14)(q13;q32) translocation that causes over-expression of the BCL-1 gene and consequent overproduction of its gene product cyclin D1. We have developed a competitive fluorescent reverse transcription polymerase chain reaction assay for the detection and semiquantitation of cyclin D1 over-expression. Using this assay a definitive ratio of the expression of cyclin D1 to cyclins D2 and D3 can be determined, provided good quality RNA is available. A single upstream primer derived from a consensus sequence found in cyclins D1, D2, and D3 was labeled at the 5' end using a fluorescent dye. Downstream primers specific to cyclins D1 and D2 were designed and used in conjunction with a previously published D3 specific primer. The fluorescently labeled PCR products were separated by electrophoresis using an ABI 377 DNA sequencer. Fluorescence emitted from each product was used to determine the ratio of expression of cyclin D1 to D2 and D3 by assigning a dosage quotient [D1/(D2+D3)]. The mean dosage quotient recorded from samples representing 29 non-MCL patients was 0.03 (SD +/- 0.03), the maximum value being 0.11. Samples from eight patients with a diagnosis of MCL generated values greater than 2. Calculation of a dosage quotient using this competitive fluorescent reverse transcription polymerase chain reaction assay allows unequivocal identification of patients with over-expression of cyclin D1, providing a new tool for the differential diagnosis of MCL.

Osteoarthritis of the hip and occupational activity.

This case-referent study investigated the relation between hip osteoarthritis and occupational activity. The subjects were 60- to 75-year-old men who had undergone intravenous urography. Interviews were obtained for 245 cases who had a hip replacement for osteoarthritis or a joint space of less than or equal to 2.5 mm and 294 referents whose joint space was greater than or equal to 3.5 mm in both hips. No clear associations were found in an analysis of all the cases, but severe disease (hip replacement for osteoarthritis or a joint space of less than or equal to 1.5 mm) was more common in the farmers, especially in those with greater than or equal to 10 years in agricultural work [odds ratio (OR) 2.0, 95% confidence interval (95% CI) 0.9-4.4]. Severe disease was also associated with prolonged standing at work (OR 2.7, 95% CI 1.0-7.3) and heavy lifting (OR 2.5, 95% CI 1.1-5.7). These associations could not be explained by obesity or sporting activity. The findings suggest an increased risk of degenerative hip disease for farmers and indicate that mechanical overloading may contribute to its pathogenesis.

Physical activity, muscle strength, and calcium intake in fracture of the proximal femur in Britain.

Regular exercise and high calcium intake possibly help to preserve bone mass. Little is known, however, about their role in preventing hip fracture. The physical activity and calcium intake of 300 elderly men and women with hip fractures were compared with those of 600 controls matched for age and sex. In both sexes increased daily activity, including standing, walking, climbing stairs, carrying, housework, and gardening protected against fracture. This was independent of other known risk factors, including body mass, cigarette smoking, and alcohol consumption. Strength of grip correlated with activity and was inversely related to the risk of fracture. Calcium intake was not related to the risk of fracture in women. Men with daily calcium intakes above 1g had lower risks. These findings point to the importance of elderly people in Britain maintaining physical activity in their day to day lives.

Dietary calcium, physical activity, and risk of hip fracture: a prospective study.

OBJECTIVE: To determine whether low dietary calcium intake and physical inactivity are risk factors for hip fracture among subjects aged 65 and over. DESIGN: Fifteen year follow up study of a large cohort of randomly selected elderly people living in the community, who had taken part in the 1973-4 survey of the Department of Health and Social Security, and for whom dietary and other data were recorded at initial interview and medical assessment. SETTING: Eight areas in Britain (England (five), Wales (one), and Scotland (two]. SUBJECTS: 1688 Subjects living in the community, of whom 1419 subjects (720 men and 699 women) agreed to participate. 1356 Subjects completed a seven day dietary record and 983 (542 men and 441 women) agreed to be assessed by a geriatrician. RESULTS: Incidence of hip fracture increased with age and was higher in women than men. Comparison with matched controls showed no evidence that the risk of hip fracture was related to calcium intake: the odds ratio for the lowest third of dietary calcium compared with the highest was 0.7 (95% confidence interval 0.1 to 3.9) after adjustment for smoking and body mass index. The adjusted odds ratio for the lowest third of outdoor activity compared with the highest was 4.3 (0.7 to 26.8), and that for grip strength was 3.9 (0.7 to 23.0). CONCLUSIONS: Reduced intake of dietary calcium does not seem to be a risk factor for hip fracture. Further evidence is provided that physical activity in the elderly protects against hip fracture.

Lytic bone disease as the presenting feature of Philadelphia-positive monosomy 7 myelodysplasia progressing to acute myeloid leukaemia.

We describe an unusual case of Philadelphia-positive, monosomy 7 myelodysplasia progressing to acute myeloid leukaemia in a 53 year old male who presented with bone-pain and B-symptoms and was found to have diffuse osteolytic lesions. Molecular genetic analysis revealed both b3a2 and e1a2 t(9;22) transcripts. Cytogenetic analysis was an additional useful test in determining the course and management of an atypical BCR-ABL positive myelodysplasia.

Large variation in t(11;14)(q13;q32) and t(14;18)(q32;q21) translocation product size is confirmed by sequence analysis of PCR products.

Polymerase chain reaction is commonly used to detect t(11;14)(q13;q32) and t(14;18)(q32;q21) chromosomal translocations associated with mantle cell lymphoma and follicular lymphoma. We tested a total of 482 samples from patients with suspected non-Hodgkin's lymphoma and sequenced unusual-sized t(11;14)(q13;q32) and t(14;18)(q32;q21) products from 33 of these patients. BCL-1 or BCL-2 gene rearrangements were confirmed in 23 of 33 patients (70%). Considerable size variation was observed using t(11;14) primers, with MTCA and MTCB t(11;14) products ranging from 234 to 934 bp and 143 to 560 bp respectively. Less variability was observed for t(14;18) Major Breakpoint Region (MBR) products (100-252 bp) but Minor Cluster Region (MCR) products ranged from 217 to 498 bp. We demonstrate the utility of sequence analysis to confirm unusual-sized translocation products and reduce false-positive results because of nonspecific amplification.

Detection of clonal T cell populations by high resolution PCR using fluorescently labelled nucleotides; evaluation using conventional LIS-SSCP.

AIMS: To detect clonal T cell populations by high resolution polymerase chain reaction (PCR) using fluorescently labelled nucleotides and analysis on an ABI 377 DNA sequencer, and to evaluate this method using low ionic strength single strand conformation polymorphism (LIS-SSCP) analysis. METHODS: DNA samples from 11 patients diagnosed with a T cell disease and 15 with no known T cell disorder were amplified using four multiplex T cell receptor gamma (TCR gamma) PCR reactions containing fluorescently labelled nucleotides. PCR products were analysed using both LIS-SSCP electrophoresis and an ABI 377 DNA sequencer using Genescan software. A Jurkat T cell leukaemia cell line was used to determine the sensitivity of the two methods. RESULTS: Clonal TCR gamma populations were detected in all 11 samples from patients with a T cell disease and no clonal populations were detected in samples from patients without a T cell disorder, using both LIS-SSCP and DNA sequencer analysis. Although the sensitivity of the two methods was comparable, the data generated by the sequencer were easier to interpret than the LIS-SSCP gels, and allowed accurate size determination of every product, which was not possible using LIS-SSCP. CONCLUSIONS: The use of fluorescent labelled nucleotides provides a more flexible and economical alternative to end labelled fluorescent primers for the detection of clonal TCR gamma gene rearrangements. This method allows clonal populations to be sized accurately and reproducibly, permitting the detection of identical clonal populations in different samples, and providing a method of monitoring disease progression and response to treatment.

Osteoarthritis of the hip and acetabular dysplasia.

The relation between acetabular dysplasia and osteoarthritis of the hip was examined in a series of 1516 pelvic radiographs taken for non-skeletal indications. Osteoarthritis was assessed by measuring joint space, and dysplasia by the centre-edge angle and acetabular depth. In contrast with previous studies of patients with symptomatic osteoarthritis of the hip, no evidence that dysplasia predisposes to osteoarthritis was found. Possible reasons for the discrepancy are discussed. It was concluded that although acetabular dysplasia may lead to osteoarthritis of the hip in some subjects, it is unlikely to be an important cause of the disease in men.

Is the hip involved in generalized osteoarthritis?

The authors examined the association of hip osteoarthritis with the presence of Heberden's nodes in a case-control study. Subjects were men aged 60-75 years who had undergone intravenous urography as out-patients. An examination of the hands was carried out in 240 patients who had a hip replacement for osteoarthritis or a joint space less than or equal to 2.5 mm, and in 290 controls whose joint space was greater than or equal to 3.5 mm in both hips. The presence of Heberden's nodes was associated with an increased risk of radiological hip osteoarthritis, the relationship being stronger in the 51 cases with severe joint-space narrowing (odds ratio = 3.4, 95% confidence interval 1.3-9.1). There was no evidence that this association differed according to the site of joint space narrowing within the hip. The finding provides further evidence that osteoarthritis of the hip can occur as part of a generalized process and does not only result from local damage to the joints.