High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Blood loss and contributing factors in femoral fracture surgery

Background

Substantial blood losses frequently accompany orthopedic procedures.

Methods

We prospectively noted peri-operative hemoglobin changes in 93 patients undergoing surgery for femoral fracture with an aim of establishing blood loss and related factors.

Results

The mean total blood loss assessed 72 hours after the surgical procedure was 3.31 (SD 1.56) units of whole blood. A multiple regression analysis revealed diathermy use and a simple fracture pattern as significant factors in reducing blood loss (p<0.01).

Conclusions

Open intramedullary fixation of femur fractures leads to considerable peri-operative blood loss. This is can be reduced by use of diathermy during surgery.     

Renal recovery after severe acute renal failure

BACKGROUND: There is limited information about renal recovery to independence from renal replacement therapy (RRT) and about factors associated with its occurrence after severe acute renal failure (ARF). METHODS: We conducted a population-based surveillance among all adult residents of the Calgary Health Region surviving ICU admission from May 1, 1999 to April 30, 2002. The primary objective was to determine the rate of and the factors associated with 90-day survival and recovery to independence from RRT in critically ill patients with severe ARF. RESULTS: At 90 days, 96 patients (40%) were alive. Of these, 72% were RRT independent with most (87%) requiring <4 weeks to recover. Prior to RRT, the median (IQR) serum creatinine and mean (SD) serum urea were 395 (252-517) micromol/L and 29.2 (18) mmol/L, respectively. Oliguria was present in 76%. Intermittent hemodialysis was the initial modality in 46% and continuous renal replacement therapy (CRRT) in 54%. By multivariate analysis, male sex (odds ratio (OR) 7.6, 95% CI, 2.2-27, p=0.01) and a diagnosis of septic shock (OR 3.9, 95% CI 1.02-14.5, p=0.05) were associated with an increased odds of recovery. Conversely, a higher Charlson co-morbidity index score (OR 0.71, 95% CI, 0.6-0.85, p=0.04) and a higher pre-RRT serum creatinine (OR 0.20, 95% CI, 0.05-0.80, p=0.02, p=0.02) were associated with reduced odds of recovery. Chronic kidney disease or the initial modality of RRT were not associated with recovery. CONCLUSIONS: The majority of severe ARF patients who survive their acute illness are independent of RRT by 90 days. Male sex and a diagnosis of septic shock are independently associated with recovery while a greater co-morbidity score and a higher serum creatinine prior to RRT are predictive of non-recovery.     

Atlanto-axial rotatory fixation: Improved demonstration using spiral CT

Atlanto-axial rotatory fixation (AARF) is an uncommon condition which is often missed at presentation because of its rarity and the relative subtlety of plain film X-ray findings, but early detection and appropriate management are vital for a cure. We describe three cases in which the use of spiral computed tomography scanning with 3D and sagittal reconstructions greatly aided diagnosis and management. The 3D images gave a more graphic picture of the overall alignment of the upper cervical spine and the skull base, while the sagittal reconstructions demonstrated the presence or absence of compensatory atlanto-occipital subluxation. The literature is briefly reviewed.     

Value of Arterialized Capillary Blood Gas Analysis in Lower Respiratory Tract Infection in Childhood

Blood gas analysis on arterialized capillary blood in 70 young infants yielded satisfactory information about oxygen therapy. Inspired oxygen concentrations above 40% are often required to raise the capillary Po₂ of hypoxic infants above 70 mmHg. Oxygen concentrations above 40% are difficult to achieve and maintain in presently available oxygen tents, though not in modern incubators, or oxygen chairs. A raised Pco₂ has frequently been found in infants with severe respiratory infections, but oxygen therapy has not resulted in CO₂ narcosis in these acute emergencies.