Biomedical subjects

Carleton T Garrett

Publications and source records attributed to Carleton T Garrett.

11 recordsLinked to original sources

PubMed2005

Data mining and clinical data repositories: Insights from a 667,000 patient data set.

Clinical repositories containing large amounts of biological, clinical, and administrative data are increasingly becoming available as health care systems integrate patient information for research and utilization objectives. To investigate the potential value of searching these databases for novel insights, we applied a new data mining approach, HealthMiner, to a large cohort of 667,000 inpatient and outpatient digital records from an academic medical system. HealthMiner approaches knowledge discovery using three unsupervised methods: CliniMiner, Predictive Analysis, and Pattern Discovery. The initial results from this study suggest that these approaches have the potential to expand research capabilities through identification of potentially novel clinical disease associations.

Clinical Chemistry Tests↗

PubMed2005

BCRABL transcript detection by quantitative real-time PCR : are correlated results possible from homebrew assays?

BACKGROUND: Quantitative real-time PCR has become the predominant molecular technique to monitor BCRABL levels in response to treatment in Ph(+) leukemia patients. However, without some form of standardized methodology between laboratories, the correlation of results is difficult. METHODS: Using TaqMan-based assays, parallel quantitative real-time PCR analysis was performed on 70 clinical specimens at Vanderbilt University Medical Center and Virginia Commonwealth University. While the same positive control cell line (K562) and quality control gene (BCR) were used, the RNA isolation technique, cDNA synthesis, BCR control cell line, and PCR primer and probe sequences were different. RESULTS: The detection of BCRABL-positive results spanned a dynamic range from 10(0) to 10(5)/100,000 cells. Forty-three samples were negative at both facilities. A Spearman rank correlation analysis was performed for the 22 BCRABL-positive paired results. The correlation coefficient, r(s), was 0.9435 (p < 0.00001), suggesting a strong correlation of the results. One discordant result was obtained for consecutive samples from one patient with a low BCRABL copy number as a result of a minimal RNA yield at one laboratory. CONCLUSIONS: These results suggest that quantitative real-time PCR assays for BCRABL detection can be comparable between laboratories despite significant differences in methodologies if the same positive control cell line and quality control gene are used. It is imperative that some level of assay standardization be adopted between laboratories, not only for patients who are monitored at different facilities, but also for larger investigative studies in which hematologic, cytogenetic and molecular responses are to be compared.

Adolescent↗

PubMed2004

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Analysis of Variance↗

PubMed2004

Evaluation of a linear amplification method for small samples used on high-density oligonucleotide microarray analysis.

High-density oligonucleotide microarray analysis has proven to be an excellent approach for gene expression profiling in human cancers. This technique assesses the expression of thousands of genes simultaneously, from at least 5 microg of total RNA per sample per experiment. This total RNA requirement poses a challenge when studying small, unique clinical samples, like biopsies. Recently, a new standardized protocol for small samples was released by Affymetrix, which includes a linear amplification step. To evaluate the impact of such amplification in the gene expression profiling of human ovarian cancer, we compared results obtained from 5 microg and 100 ng of total RNA from the same tumor sample, using the standard Affymetrix protocol and the new linear RNA amplification protocol, respectively. We identified a small bias in gene expression data caused by linear amplification, potentially due to shorter elongation products leading to misclassification of probe sets directed to the middle-5' region of the transcripts. Interestingly, the magnitude of the bias varied when different normalization and expression summary algorithms were used. However, this bias does not affect tumor gene expression profiling. Consequently, linear amplification may be of utility in cases of extremely low RNA recovery from critical and unique samples, such as small biopsies.

Cluster Analysis↗

PubMed2003

Hepatic artery thrombosis after liver transplantation and genetic factors: prothrombin G20210A polymorphism.

Hepatic artery thrombosis (HAT) after liver transplantation is associated with a high incidence of graft failure. The incidence ranges between 2% and 25%, with an overall incidence of approximately 7%. Different risk factors have been associated, but the participation of genetic factors in the cause of HAT is less well studied. A single-base change (G to A) at position 20210 in the 3' untranslated region of the prothrombin gene is associated with increased plasma levels of prothrombin and might therefore increase the risk for thrombosis. We reviewed our HAT experience in 11 years at Medical College of Virginia hospitals of 491 patients undergoing 533 liver transplantations. There were 14 liver grafts with documented HAT (2.62%) in 13 patients. Prothrombin G20210A polymorphism was found in the DNA obtained from 2 of 14 liver allograft tissues (14.2%) but not in the DNA from leukocytes obtained from the peripheral blood of recipients with HAT.

Amino Acid Substitution↗

PubMed2003

Effects of stress management on PNI-based outcomes in persons with HIV disease.

A pretest-posttest, repeated-measures design was used to evaluate the effects of two stress management interventions on a battery of outcomes derived from a psychoneuroimmunological (PNI) framework. The effects of cognitive-behavioral relaxation training groups (CBSM) and social support groups (SSG) were compared with a WAIT-listed control group on the outcomes of psychosocial functioning, quality of life, neuroendocrine mediation, and somatic health. Participants were 148 individuals (119 men, 29 women), diagnosed with HIV disease; 112 (76%) completing the study groups. Using analysis of covariance, the CBSM group was found to have significantly higher postintervention emotional well-being and total quality-of-life scores than did either the SSG or WAIT groups. SSG participants had significantly lower social/family well-being scores immediately postintervention and lower social support scores after 6 months. The findings point to a pressing need for further, well-controlled research with these common intervention modalities.

Adaptation, Psychological↗

PubMed2003

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology.

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.

Adult↗

PubMed2003

Comparison of three commercially available assays for HCV RNA using the international unit standard: implications for management of patients with chronic hepatitis C virus infection in clinical practice.

OBJECTIVES: The present study was performed to evaluate the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. METHODS: The three assays used were Amplicor Monitor PCR, the National Genetics Institute PCR assay, and branched chain DNA. HCV RNA was measured at four time points (baseline, 3 months after the start of therapy, at the end of treatment, and 6 months after discontinuation of therapy) in 106 consecutive patients who received interferon and ribavirin for chronic HCV. RESULTS: The mean age of the patients was 44 yr. Of the patients, 62% were male, 24% were African American, 38% had bridging fibrosis or cirrhosis, and 75% were HCV genotype 1. Of the 424 samples analyzed, 82-89% of values were within 1 log unit and 85-92% were within 2 log units by the various assays. This variability was not dependent upon HCV genotype. HCV RNA was undetectable in 1.4-6.8% of samples when virus was detected by another assay. The mean HCV RNA in these discordant samples was 1.47-6.33 log IU/ml (30-2,100,000 IU/ml). CONCLUSIONS: These data demonstrate that approximately 90% of serum values for HCV RNA were within 1 log unit by the international unit standard regardless of which virological assay was used. However, false positive and false negative results as well as variations in the HCV RNA level of more than 1 to 2 log units can occur with any of the assays, and these results may have an impact upon the management of patients receiving interferon therapy. It is therefore unwise in clinical practice to base important treatment decisions upon a single HCV RNA determination.

Adult↗

PubMed2002

Analytical validation of a real-time reverse transcription-polymerase chain reaction quantitation of different transcripts of the Wilms' tumor suppressor gene (WT1).

Transcript variants of the same gene may play distinct functions in the tissue where they are expressed. Absolute quantitation of different transcript variants in malignant and normal tissues can address the specific role of each particular isoform in cancer development and progression. We have recently demonstrated differential expression of the wild-type Wilms' tumor transcript (wtWT1) and a novel truncated WT1 transcript (trWT1) which lacks the first five exons of wtWT1, among human prostate cancer, leukemia, and breast cancer cell lines. Here we report the analytical validation of a real-time RT-PCR assay for the absolute quantitation of these two different WT1 transcripts with specific primers and probes that ensure specificity for each WT1 variant. By cloning each WT1 transcript in a T3 promoter-containing plasmid, we obtained two WT1 transcript-specific in vitro-generated RNA calibrators for absolute quantitation. Serial dilution of each RNA calibrator demonstrated a 5 log linear dynamic range (5 x 10(1) to 5 x 10(6) copies/reaction, R(2)=0.9963 for wtWT1 and R(2)=0.9993 for trWT1). Dilution of the calibrators in total RNA from 1 x 10(3) non-WT1-expressing cells showed a decreased sensitivity without affecting the linear dynamic range. Precision studies for values within the linear dynamic range showed a coefficient of variation of less than 4% for both transcripts. The described method provides a sensitive and reliable technique for quantitating different WT1 mRNA transcripts.

Calibration↗

PubMed2002

Human immunodeficiency virus genotype and hypertriglyceridemia.

Many HIV patients develop a progressive syndrome of abnormal body fat distribution accompanied by hypertriglyceridemia. Antiretroviral agents are thought to be etiologic in the syndrome, often termed "highly active antiretroviral therapy (HAART)-associated lipodystrophy." In the course of clinical HIV genotype testing, we observed that our HIV patients with hypertriglyceridemia had viral genotypes that were more highly mutated than those of our therapy-matched control patients. Hypertriglyceridemia was statistically associated with predicted resistance for three nucleoside reverse transcriptase inhibitors: zidovudine, abacavir, and stavudine. Statistical analysis of 51 patients in retrospect revealed a strong association of mutations at reverse transcriptase codons M41 and T215 with hypertriglyceridemia (chi-square (chi(2)) = 8.375, P=.0038; and chi(2)=7.445, P=.0064, respectively). This was in contrast to silent mutations, which occurred at equivalent rates in retroviral genotypes of patients with and without hypertriglyceridemia. The findings imply that the HIV genotype itself may be a significant etiologic factor in antiretroviral-associated lipodystrophy.

Adult↗

PubMed2002

Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.

Breast Neoplasms↗