Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium.

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays.

Absorption, tissue distribution, metabolism and elimination of taurine given orally to rats.

Three biodisposition studies with taurine were performed in male and female adult rats at dosages of 30 and 300 mg/kg. A single oral dose of (14)C-taurine was rapidly absorbed, distributed to tissues and excreted unchanged in urine. Elimination of radioactivity from intracellular pools was slow. Pre-treatment of animals for 14 days with unlabelled taurine did not significantly affect the fate of (14)C-taurine. At the higher dose there was more extensive excretion combined with a lower percentage of the dose in the carcass, indicating the possibility of saturation of the tubular reabsorption mechanism for taurine. Daily administration of unlabelled taurine for 14 days did not result in an increase in total taurine in the brain. The data indicate that exogenous taurine rapidly equilibrates with endogenous body pools and that any excess is rapidly eliminated by the kidneys.

Efficacy of infrainguinal bypass for limb salvage in young diabetic patients.

The efficacy of infrainguinal bypass for limb salvage in young diabetic patients has not been well established. The purpose of this study is to determine the intermediate-term results (patency and limb salvage) of infrainguinal revascularization carried out for limb salvage (rest pain or ulceration) in young (<50 years old) diabetic atherosclerotic patients. Thirty-nine bypasses in 31 patients with a mean age of 44 years were retrospectively reviewed. There were no perioperative deaths. Minor or major complications occurred in 23% of cases. By life table analysis, the 18-month primary patency rate was 60+/-11%, assisted primary patency rate was 78+/-9%, and limb salvage rate was 71+/-9%. Most major amputations (five of nine) were required in patients with functional bypasses, either because of persistent infection or failure of wound healing. The presence of severe stenoses (>70%) in all three major named foot vessels (dorsalis pedis, medial and lateral plantar arteries) was associated with a high likelihood of limb loss despite a patent bypass (p<0.05). We could not identify any other factors statistically predictive of thrombosis, amputation, or the need for graft revision. Infrainguinal revascularization in this patient population can be carried out with acceptable limb salvage rates. However, patients should be made aware of the high incidence of amputation regardless of the success of the revascularization procedure, particularly in the presence of severe occlusive disease within the foot.

Vascular evaluation of the problem diabetic foot.

Ischemia plays a pivotal role in the management of the problem diabetic foot. Prompt revascularization offers the patient with diabetes with lower-extremity ischemia the best hope for limb salvage and normal ambulation. The true vascular status of the diabetic foot may be difficult to assess by clinical examination. Because of the dangers of missing correctable vascular disease, noninvasive vascular testing plays an important role in the evaluation of the problem diabetic foot. The laboratory should have documented reliability, and its results must be interpreted in the context of the patient's clinical progress. The most common laboratory error is overestimating the blood supply to the foot because of technical problems with mural calcification. Algorithms for the use of the vascular laboratory for common foot problems are presented. The vascular laboratory, although helpful, is no substitute for clinical judgement. When ischemia is suspected or when response to conservative care is poor, early vascular surgical consultation is prudent.

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.

Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction.

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.

Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection.

The development of technology to increase the sensitivity and speed of detection of bacterial pathogens in samples is important for diagnosis and monitoring of illness. We have developed a sensitive and rapid method for the detection of bacteria, using Escherichia coli as a model, which combines transcription-based target amplification with a bead-based sandwich hybridization assay using rare earth metal chelate labelled probes and time-resolved fluorescence detection. Using these methods as little as 100 copies (0.00016 attomoles) of purified native Escherichia coli rRNA or just one bacterial cell in a spiked sample could be detected. These results demonstrate that amplification of rRNA by transcription-based amplification and detection by time-resolved fluorescence provide a sensitive technology for the direct detection of micro-organisms without the requirement for prior cultivation.

Rapid antimicrobial susceptibility testing of gram-negative bacilli using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A.

The MicroScan Rapid Neg MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of Gram-negative bacilli. In a three site clinical study eleven antimicrobial agents were evaluated by comparing results obtained with 741 clinical isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels determined visually. Results for 31%, 40%, 12% and 9% of the isolates were available within 3.5, 4.5, 5.5 and 7.0 hours respectively. Results for 7.3% were not available within that time period. For the seven drugs analyzed using a Minimum Inhibitory Concentration range of dilutions, overall agreement (+/- 1 dilution) was 94%, with 1.5% very major, 0.9% major and 2.5% minor errors. For the four drugs analyzed using a Breakpoint range of dilutions, overall agreement (+/- 1 dilution) was 97%, with two percent very major, and one percent major errors. The MicroScan Rapid Neg MIC system is an accurate and rapid method for same day determination of susceptibility of Gram-negative bacilli.

Rapid antimicrobial susceptibility testing of gram-positive cocci using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A.

The Microscan Rapid Pos MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of staphylococci, streptococci, and Listeria. In a three site clinical study, panels containing 26 antimicrobial agents were evaluated by comparing results obtained with 605 clinically significant isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels. Results for 16%, 40%, 13%, 9%, 8%, 11% and 1% of the isolates were available within 3.5, 4.5, 5.5, 7.0, 8, 11 and 15 h respectively. Results for 2% were not available within that time period. Overall agreement (+/- 1 dilution) for the 14,609 efficacy comparisons was 96%, with 1% each for very major, major and minor errors. Interlaboratory reproducibility testing of 25 isolates in triplicate in each site, showed an overall essential agreement of 97%. The MicroScan Rapid Pos MIC System is an accurate, reproducible and rapid method for same-day determination of susceptibility of Gram-positive cocci.

Rapid identification of Enterobacteriaceae with microbial enzyme activity profiles.

A total of 539 clinical isolates belonging to 10 species of the Enterobacteriaceae family were identified by enzyme activity profiles within 30 min of test inoculation. Each isolate was grown at 37 degrees C for 18 h on Mueller-Hinton agar and suspended to an optical density of 200 Klett units on 0.85% saline. Enzyme activity profiles were obtained by inoculating 18 fluorogenic substrates with the standardized bacterial suspension and monitoring initial rates of hydrolysis over the first 30 min of analysis. Individual enzyme activity profiles were entered into a coded data bank, and identifications were based on the Bayesian theory of probabilities. At a confidence level of 95%, five species were identified with a greater than 90% efficiency, three species were identified between 83 and 88% efficiency, and two species demonstrated a 72 and 75% efficiency of identification. The enzyme activity profile method of bacterial identification is rapid, easily automated, and reproducible.