PubMed Health⌕ Search

Biomedical subjects

T E Mifflin

Publications and source records attributed to T E Mifflin.

At least 19 recordsLinked to original sources
PubMed2000

Robotic automation performs a nested RT-PCR analysis for HCV without introducing sample contamination.

The Polymerase Chain Reaction (PCR) is a popular method to amplify and detect specific RNA and DNA sequences. To obtain maximum performance of PCR, it is best performed by highly skilled technologists because of the complexity of the assay and the potential for laboratory contamination from the amplification products produced. We chose to automate this nested RT-PCR for hepatitis C assay to significantly reduce the need for manual pipetting while preserving the excellent non-contamination performance of the corresponding manual test. A three axis cartesian robotic pipetting station was equipped to perform RT-PCR using an on-board automated thermal cycling device. 104 sera were analyzed using this modified pipetting station and we found a very close agreement (100% sensitivity and 98% specificity) with results previously obtained by corresponding manual RT-PCR analysis. This study demonstrated a user-programmed robotic pipetting system could successfully automate a complex PCR assay without contamination. Our results suggest that use of robotic pipetting station can provide cost efficient alternative to performance of molecular diagnostic assays while demonstrating minimal inter sample contamination.

Electrophoresis, Agar Gel↗
PubMed1996

Gaucher disease: studies of phenotype, molecular diagnosis and treatment.

This report summarizes the results on 39 patients with Gaucher disease who have been genotyped, evaluated, and/or followed at this center. Mutation analysis for 4 common mutations; N370S, L444P, 84gg and IVS2 (+1), was performed for all patients. Mutation analysis identified both mutant alleles in 69% and at least one mutant allele in 90% of all chromosomes. This study group of 39 patients included 32 type 1, four type 2 and three type 3 patients. We include the details of the clinical course of two patients with Gaucher disease treated with enzyme replacement therapy (ERT). One patient with chronic neuronopathic Gaucher disease has been treated with enzyme replacement therapy (ERT) at a dose of 60 U/kg every 2 weeks since 2.5 years of age and has shown no progression of neurologic involvement. A second patient with non-neuronopathic Gaucher disease has demonstrated an unusually delayed response to ERT. No clinical response was noted following 17 months of treatment at 60 U/kg every 2 weeks. Only after the dose was increased to 60 U/kg every week was a clinical response evident. Response to treatment at 15 U/kg every 2 weeks was variable in the four type 1 patients treated at the lower dose. In two of these patients with identical genotypes, one patient demonstrated a positive clinical response to low dose treatment while the other patient did not.

Adolescent↗
PubMed1995

Molecular diagnosis of cystic fibrosis.

The clinical and pathologic features of patients with cystic fibrosis are summarized, and generalized genotype or phenotype correlations are discussed in this article. Incorporation of modern molecular biologic techniques into a rapid, cost-efficient, and specific diagnostic laboratory test is outlined. The protocol for the multiplex polymerase chain reaction detection of five common cystic fibrosis transmembrane conductance regulator (CFTR) mutations by ASO hybridization is detailed. Neonatal screening and issues involved in the genetic counseling of families at risk for cystic fibrosis are presented. Recommendations for molecular diagnostic testing in cystic fibrosis are made.

Base Sequence↗
PubMed1995

Enzymatic and molecular diagnosis of Gaucher disease.

Advances in the knowledge of the molecular genetics of Gaucher disease has made diagnosis more certain. Carrier detection in kindreds in which the responsible mutation has been defined is completely reliable now. Coupled with enzymatic assays, the diagnostic capabilities are greater than before. Use of these methods provides important information to individuals at risk and allows them to make critical decisions. The new, simplified methods reviewed in this article permit the molecular diagnosis of the disease and carrier stage of large numbers of samples within 1 week.

Base Sequence↗
PubMed1991

Localization of rhinovirus replication in vitro with in situ hybridization.

To facilitate understanding of human rhinovirus (HRV) pathogenesis, methods were developed for detection of HRV infection in vitro using in situ hybridization (ISH). HRV-14 RNA probes and oligonucleotide probes representing conserved sequences in the 5'-non-translated region were labeled with 35S and used to detect infected HeLa or WI-38 strain human embryonic lung cells in cytological preparations. ISH was shown to be specific for detection of HRV on a single-cell basis. Subsequently, in human nasal polyps infected in vitro, both oligonucleotide- and ribo-probes produced a strong signal in association with ciliated epithelial cells. In human adenoids infected in vitro, a signal was observed in non-ciliated epithelial cells. This study shows that HRV replicates in ciliated cells in the epithelium of human nasal polyps infected in vitro, and the presence of viral RNA in non-ciliated cells of the human adenoid infected in vitro suggests that other cell types may also support rhinovirus replication.

Adenoids↗
PubMed1989

Pancreatic amylase measured in serum by use of a monoclonal antibody immunochemically immobilized to a solid phase.

We studied a method for measuring the pancreatic isoenzyme of amylase (EC 3.2.1.1) by use of a mouse monoclonal antibody against human salivary-type amylase (Clin Chem 1985;31:1283) coupled indirectly to particles of polyvinylidene fluoride via polyclonal goat anti-mouse immunoglobulin. These particles, in 200 microL of a suspension, could remove salivary amylase (activity 2200 U/L) from an equal volume of serum in 5 min. Measurement of amylase activity in the supernatant fluids from treated sera thus provided an assay of pancreatic amylase. Precision studies at three activity concentrations yielded within-run CVs of 1.6% to 1.7% (n = 25) and total CVs of 2.2% to 5.1% (20 days). Salivary amylase added to each of 10 sera was completely (99.8%, SD 1.6%) removed. The new method (y) showed the following regression statistics when compared with an electrophoretic method (x): slope = 0.989 (SD 0.019), intercept = -0.220% (SD 1.48%), SEE 4.0%, n = 51. Similar respective regression values were found for urine samples: slope = 0.934 (SD 0.053), intercept = 2.3 U/L (SD 3.2), SEE 8.4 U/L, n = 26. The following respective values were found when the new method (y) was compared with the previously described immunoprecipitation assay (x): slope = 1.02 (SD 0.02), intercept = 2.2% (SD 1.4%), SEE 3.3%, n = 23 sera. Reference intervals for pancreatic amylase activity in serum were established for three different substrates: maltotetraose, maltopentaose, and p-nitrophenylheptaoside.

Acute Disease↗
PubMed1989

Interaction of immobilized anti-salivary amylase antibody with human macroamylases: implications for use in a pancreatic amylase assay to distinguish macroamylasemia from acute pancreatitis.

We examined the ability of an immobilized antibody to salivary amylase (Clin Chem 1985;33:1283-8) to react with amylase in macroamylasemic sera. The antibody removed 50% (SD 23%) of the total amylase activity from 39 macroamylase sera, a percentage indistinguishable (P greater than 0.75) from the percentage removed from concurrently analyzed sera from healthy volunteers (49%, SD 11%). Electrophoretic analysis of 23 macroamylasemic sera revealed that the antibody removed only part of the macroamylase band(s) in 71% of the cases. We conclude that the mean isoenzyme composition of the macroamylase complexes is essentially identical to the mean isoenzyme distribution in normal sera (i.e., about half salivary and half pancreatic amylase). Further, the immobilized antibody can be used to distinguish most patients with macroamylasemia from those with acute pancreatitis, because sera from the latter contain an increased proportion (greater than 80%) of pancreatic amylase.

Acute Disease↗
PubMed1989

Use and applications of nucleic acid probes in the clinical laboratory.

The use and availability of nucleic acid probes are significantly greater than they were three to five years ago. This increased utilization has resulted because of numerous changes in molecular genetic procedures. First, the sources of probes have shifted from cloned genomic and cDNA fragments to single-strand oligonucleotides and RNA or "riboprobes." These newer probes offer defined composition and capability to create separate sense and anti-sense strands. Second, an increased variety of labeling techniques provide more ways to incorporate isotopic and nonisotopic species into these nucleic acid fragments than before. Third, the advent of nucleic acid amplification forecasts the ability to detect sub-attomol quantities of target nucleic acids, which could not be detected by classical hybridization methods. Fourth, more-sensitive signaling systems (e.g., chemiluminescence) now available will also contribute to the lowering of detection limits. Fifth, alternative sample-processing methods will shorten the time required to perform these assays. Finally, coupling these newer molecular probe procedures with automation will lead to improved precision and reliability and promote their introduction into the clinical laboratory as routine procedures. Additional factors such as clinical correlation, cost analysis, etc. will stimulate clinical laboratory personnel to participate further in development and use of these procedures.

Clinical Laboratory Techniques↗
PubMed1987

Comparison of radioactive (32P and 35S) and biotinylated probes for detection of cytomegalovirus DNA.

Cytomegalovirus (CMV) DNA was detected in a dot-blot assay by hybridization to a DNA probe labeled with radioisotopes (32P or 35S) or biotin. Limits of detection were established for both the radioisotopically labeled DNA probes as well as the biotin-labeled probe. Hybridization of the radioisotopically labeled probes was detected by autoradiography and liquid scintillation while the biotin-labeled probe was detected after coupling to one of three enzymes (e.g., horseradish peroxidase, alkaline phosphatase, or acid phosphatase). In addition, several different substrates were evaluated with the nonisotopic detection enzymes. Detection limits (and times for detection) were 1 pg (4 h) for 32P, approximately 1 pg (96 h) for 35S, 5 pg (1-3 h) for the phosphatases, and 25-50 pg for peroxidase. Thus, 32P-labeled probes appear to provide the best sensitivity whereas the avidin-linked phosphatases provide the best sensitivity among the nonisotopic detection systems.

Autoradiography↗
PubMed1987

Serum amylases in chronic and end-stage renal failure: effects of mode of therapy, race, diabetes and peritonitis.

Serum total amylase, pancreatic amylase and lipase activities were studied prospectively in 43 hemodialysis, 22 peritoneal dialysis and 22 chronic renal failure patients. None of the patients had symptoms of pancreatic disease at the time of study. Mean total amylase activities were similar and above the upper limit of normal in the 3 treatment groups. Total amylase was abnormal in 75% of the patients and exceeded twice the upper limit of normal in 24%. Blacks and nondiabetics had higher levels than whites and diabetic patients, respectively. Percentage pancreatic amylase exceeded the upper limits of normal in one third of the patients. Mean pancreatic amylase was above the upper normal limit in the 3 groups, and values were abnormal in 63% of all patients. Mean pancreatic amylase activity was significantly lower in peritoneal dialysis than in hemodialysis or chronic renal failure patients (p = 0.01). Pancreatic amylase activity was unaffected by race. The higher total amylase activity in blacks was due to increased salivary isoenzyme. Hemodialysis treatments did not change total amylase or pancreatic amylase activity. Mean lipase activity approximated the upper limit of normal in the 3 groups and values were abnormal in 42% of all patients. Serum total amylase and pancreatic amylase activity did not increase during episodes of peritonitis in the peritoneal dialysis group. Peritoneal dialysis, whether or not accompanied by peritonitis, was responsible for removal of only a small amount of amylase activity per day.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1986

Methotrexate interferes with determinations of conjugated bilirubin with the Kodak Ektachem 400.

Immediately after intravenous infusion of a high dose (concentration in serum greater than 1000 mumol/L) of methotrexate, the apparent conjugated bilirubin (Bc) concentrations in serum of two osteosarcoma patients, as measured by the Kodak Ektachem 400 analyzer, were greater than the corresponding total bilirubin concentrations, but decreased as the concentrations of methotrexate in serum decreased. In an interference study we found that methotrexate added to sera containing a wide range of basal Bc concentrations increased the measured Bc concentration in a linear and dose-related fashion. Methotrexate also interfered negatively with measurements of unconjugated bilirubin (Bu). The source of the interference appears to be an overlap in the absorption spectrum of methotrexate with Bc and Bu at 400 nm.

Autoanalysis↗
PubMed1986

An optimized method for measuring cyclosporin A with 125I-labeled cyclosporin.

We evaluated the use of the new iodinated ligand for the in vitro measurement of cyclosporin A by radioimmunoassay (RIA). Substitution of the iodinated cyclosporin (125I-CyA) for the corresponding tritium-labeled analog (3H-CyA) considerably simplifies and accelerates the currently available RIA, and improves its precision. Analysis of the respective dose-response curves showed that the 50% B0 value was lower for the 125I-CyA assay than for the 3H-CyA assay (37 vs 77 micrograms/L). Use of whole-blood specimens minimized interferences from temperature and hematocrit. We conclude that the use of 125I-CyA in a commercially available RIA for whole-blood specimens is accessible to most laboratories and provides rapid, reproducible data for management of transplant patients.

Computers↗
PubMed1986

Electrophoretic assays of amylase isoenzymes and isoforms.

Electrophoretic methods provide an effective means for analysis of amylase isoenzymes and isoforms. A wide variety of such techniques have been developed and reflect different applications and analytic objectives of different investigators. Virtually all of the available clinical data on amylase isoenzymes and isoforms has resulted from application of these methods. Recent identification of selective amylase inhibitors and of monoclonal antibodies to amylase isoenzymes holds promise of development of rapid routine clinical assays for amylase isoenzymes. Electrophoretic methods will have an important role in validating these new assays. Furthermore, at the present time, electrophoretic methods provide the best approach for analyzing isoforms of the pancreatic and salivary isoenzymes. This may be clinically important in patients with tumors, pancreatic pseudocysts, or macroamylasemia. Further clinical experience is needed to define the roles of the various assays that are now available for the estimation of amylase isoenzymes and isoforms in human samples.

Blood Protein Electrophoresis↗