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PubMed1998

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines.

The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories.

Alleles↗
PubMed1991

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.

Anti-Bacterial Agents↗
PubMed1991

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.

Anti-Bacterial Agents↗
PubMedDate not supplied

Optimization, assessment, and proposed use of a direct nested reverse transcription-polymerase chain reaction protocol for the detection of hepatitis C virus.

OBJECTIVES: To compare the performance of reverse transcription followed by the polymerase chain reaction (RT-PCR), without RNA purification, with the performance of classic protocols. STUDY DESIGN/METHODS: Direct and classic techniques were used to test three groups of samples: six hepatitis C virus (HCV) seroconversion panels (n = 90), a HCV RNA reference panel (n = 26), and serial dilutions of four HCV-positive sera (n = 24). These methods were then applied sequentially through a clinical diagnostic algorithm to test 268 samples from high-risk patients. RESULTS: For the three groups of samples, we found a 94% concordance between direct and purified RT-PCR methods. For the detection of HCV RNA in clinical samples, sensitivity was maximized and cost minimized using both protocols according to the proposed algorithm. CONCLUSIONS: The direct PCR method is reliable, sensitive, and can result in time and cost savings. The suggested testing algorithm can enhance sensitivity and time savings for populations with a high prevalence of infection.

Algorithms↗
PubMedDate not supplied

[A modification in technology improves the specificity of the Du Pont de Nemours immunoenzyme kit for the detection of anti-HIV antibodies].

Du Pont De Nemours perfected a new enzyme immunoassay for the screening of anti-HIV 1 antibodies, characterized by its rapidity: 2 hours of incubation, when the normal procedure needed 3 hours 30 minutes of incubation. We tested with the normal procedure and the shortened one 4,025 randomly selected blood donors. 55 samples of the SNTS panel and 10 sera representing the Western-Blot reference panel of the "Retrovirus" study group of the SNTS. We can say that shortening the incubation time results in certain advantages opposite the previous procedure: time saving (1 hour 30 minutes); improved specificity: the rate of false positive, 0.37% with the normal method, drops to 0.07% with the short one; good sensitivity, since all the individual positive samples were detected but it seems slightly less sensitive than the normal method for the diluted positive samples.

Antibodies, Viral↗
PubMedDate not supplied

[Use of monoclonal antibodies for studying the classical hog cholera virus].

Numerous monoclonal antibodies (MAb) to hog cholera virus are a highly specific and effective instrument for studies of this agent. Panels of MAb for differential diagnosis of Pestiviruses are characterized. International reference panel of 30 MAbs is a result of cooperation of European scientists; it was approved as the official reference for assessing all available and new diagnostic agents. MAb permit intraspecies differentiation between hog cholera virus strains and, which is particularly important, between vaccine and field strains. Study of antigenic structure and functional characteristics of surface proteins of the virus with the use of MAb panels helped single out and map the functions of four antigenic sites of surface glycoprotein E2 and detect a relationship between RNAse activity and structural component of another surface glycoprotein E0.

Animals↗
PubMed2006

How well do HapMap SNPs capture the untyped SNPs?

BACKGROUND: The recent advancement in human genome sequencing and genotyping has revealed millions of single nucleotide polymorphisms (SNP) which determine the variation among human beings. One of the particular important projects is The International HapMap Project which provides the catalogue of human genetic variation for disease association studies. In this paper, we analyzed the genotype data in HapMap project by using National Institute of Environmental Health Sciences Environmental Genome Project (NIEHS EGP) SNPs. We first determine whether the HapMap data are transferable to the NIEHS data. Then, we study how well the HapMap SNPs capture the untyped SNPs in the region. Finally, we provide general guidelines for determining whether the SNPs chosen from HapMap may be able to capture most of the untyped SNPs. RESULTS: Our analysis shows that HapMap data are not robust enough to capture the untyped variants for most of the human genes. The performance of SNPs for European and Asian samples are marginal in capturing the untyped variants, i.e. approximately 55%. Expectedly, the SNPs from HapMap YRI panel can only capture approximately 30% of the variants. Although the overall performance is low, however, the SNPs for some genes perform very well and are able to capture most of the variants along the gene. This is observed in the European and Asian panel, but not in African panel. Through observation, we concluded that in order to have a well covered SNPs reference panel, the SNPs density and the association among reference SNPs are important to estimate the robustness of the chosen SNPs. CONCLUSION: We have analyzed the coverage of HapMap SNPs using NIEHS EGP data. The results show that HapMap SNPs are transferable to the NIEHS SNPs. However, HapMap SNPs cannot capture some of the untyped SNPs and therefore resequencing may be needed to uncover more SNPs in the missing region.

Asian People↗
PubMed1995

Functional assessment of CD2, CD3 and CD28 on the surface of peripheral blood T-cells from infants at low versus high genetic risk for atopy.

Recent studies from several laboratories suggest that the rate of postnatal maturation of T-cell function(s) associated with in vitro activation may be slower in children at high genetic risk for atopy (HR), compared to their normal (low risk; LR) counterparts. The present study compared the in vitro activity of the function-associated surface molecules CD2, CD3 and CD28 in panels of 27 HR and 13 LR infants, with a reference panel of 10 adults, employing assay systems involving T-cell stimulation with MoAbs against these molecules. The response maxima induced by saturating levels of the MoAbs were equivalent in all 3 groups, but T-cells from the HR infants required 10-50 fold higher levels of anti-CD3 stimulation to attain their maximum response, relative to adults (p = 0.02); T-cells from LR infants were also less responsive to anti-CD3 than adults, but these differences were smaller and did not attain statistical significance. It is suggested that these differences are attributable to varying proportions of competent T-memory cells (which respond to low levels of anti-CD3) in PBL from these populations, the postnatal accumulation of which proceeds slowest in the HR group.

Adult↗
PubMed1993

[An analysis of the activities of the United Register of Typed Donors and of the status of tissue typing in different regions of Russia].

The paper presents a review of the activities of the National Register of Typed Donors in connection with 1.5-year activity of the World Donor of Bone Marrow Association. The search for donors was performed for all European and American countries using myelotransformation from unrelated donor. Basing on I class HLA-antigen phenotyping frequency, split typing and DR-antigens, conclusions are drawn as to the condition of tissue typing by the register as a whole and for separate regions of the country. The composition and contamination with nonmonospecific sera of the National Panel of Typing Sera have been reviewed in comparison with reference panel one lambda.

Blood Transfusion↗
PubMed2006

Detecting disease-causing mutations in the human genome by haplotype matching.

Comparisons between haplotypes from affected patients and the human reference genome are frequently used to identify candidates for disease-causing mutations, even though these alignments are expected to reveal a high level of background neutral polymorphism. This limits the scope of genetic studies to relatively small genomic intervals, because current methods for distinguishing potential causal mutations from neutral variation are inefficient. Here we describe a new strategy for detecting mutations that is based on comparing affected haplotypes with closely matched control sequences from healthy individuals, rather than with the human reference genome. We use theory, simulation, and a real data set to show that this approach is expected to reduce the number of sequence variants that must be subjected to follow-up analysis by at least a factor of 20 when closely matched control sequences are selected from a reference panel with as few as 100 control genomes. We also define a reference data resource that would allow efficient application of this strategy to large critical intervals across the genome.

Alleles↗
PubMed2004

Extended microsatellite analysis in microsatellite stable, MSH2 and MLH1 mutation-negative HNPCC patients: genetic reclassification and correlation with clinical features.

BACKGROUND: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder predisposing to predominantly colorectal cancer (CRC) and endometrial cancer frequently due to germline mutations in DNA mismatch repair (MMR) genes, mainly MLH1, MSH2 and also MSH6 in families seen to demonstrate an excess of endometrial cancer. As a consequence, tumors in HNPCC reveal alterations in the length of simple repetitive genomic sequences like poly-A, poly-T, CA or GT repeats (microsatellites) in at least 90% of the cases. AIM OF THE STUDY: The study cohort consisted of 25 HNPCC index patients (19 Amsterdam positive, 6 Bethesda positive) who revealed a microsatellite stable (MSS)--or low instable (MSI-L)--tumor phenotype with negative mutation analysis for the MMR genes MLH1 and MSH2. An extended marker panel (BAT40, D10S197, D13S153, D18S58, MYCL1) was analyzed for the tumors of these patients with regard to three aspects. First, to reconfirm the MSI-L phenotype found by the standard panel; second, to find minor MSIs which might point towards an MSH6 mutation, and third, to reconfirm the MSS status of hereditary tumors. The reconfirmation of the MSS status of tumors not caused by mutations in the MMR genes should allow one to define another entity of hereditary CRC. Their clinical features were compared with those of 150 patients with sporadic CRCs. RESULTS: In this way, 17 MSS and 8 MSI-L tumors were reclassified as 5 MSS, 18 MSI-L and even 2 MSI-H (high instability) tumors, the last being seen to demonstrate at least 4 instable markers out of 10. Among all family members, 87 malignancies were documented. The mean age of onset for CRCs was the lowest in the MSI-H-phenotyped patients with 40.5 +/- 4.9 years (vs. 47.0 +/- 14.6 and 49.8 +/- 11.9 years in MSI-L- and MSS-phenotyped patients, respectively). The percentage of CRC was the highest in families with MSS-phenotyped tumors (88%), followed by MSI-L-phenotyped (78%) and then by MSI-H-phenotyped (67%) tumors. MSS tumors were preferentially localized in the distal colon supposing a similar biologic behavior like sporadic CRC. MSH6 mutation analysis for the MSI-L and MSI-H patients revealed one truncating mutation for a patient initially with an MSS tumor, which was reclassified as MSI-L by analyzing the extended marker panel. CONCLUSION: Extended microsatellite analysis serves to evaluate the sensitivity of the reference panel for HNPCC detection and permits phenotype confirmation or upgrading. Additionally, it confirms the MSS status of hereditary CRCs not caused by the common mutations in the MMR genes and provides hints to another entity of hereditary CRC.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Detection of microsatellite instability from archival, hematoxylin-eosin-stained colorectal cancer specimen.

Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer (HNPCC). Owing to early onset of colorectal cancer before the age of 50 years and/or familial clustering of HNPCC-related malignancies, the diagnosis of HNPCC was suspected in 2 patients. Because no paraffin-embedded tumor tissue was available, we used archival 5-microm, hematoxylin-eosin-stained tumor specimen slides for direct MSI analysis. Tissue was microdissected and cells were lysed using 1% Triton. Fluorescence polymerase chain reaction amplification of a panel of 7 microsatellite markers, including all markers of the current international reference panel (BAT-25, BAT-26, D2S123, D5S346, and D17S250), demonstrated MSI in one patient and excluded MSI in the other. In conclusion, this study demonstrates the feasibility of MSI analysis by direct fluorescence polymerase chain reaction amplification using hematoxylin-eosin-stained tissue specimens without the need for prior DNA extraction.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed1998

A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.

In December 1997, the National Cancer Institute sponsored "The International Workshop on Microsatellite Instability and RER Phenotypes in Cancer Detection and Familial Predisposition," to review and unify the field. The following recommendations were endorsed at the workshop. (a) The form of genomic instability associated with defective DNA mismatch repair in tumors is to be called microsatellite instability (MSI). (b) A panel of five microsatellites has been validated and is recommended as a reference panel for future research in the field. Tumors may be characterized on the basis of: high-frequency MSI (MSI-H), if two or more of the five markers show instability (i.e., have insertion/deletion mutations), and low-frequency MSI (MSI-L), if only one of the five markers shows instability. The distinction between microsatellite stable (MSS) and low frequency MSI (MSI-L) can only be accomplished if a greater number of markers is utilized. (c) A unique clinical and pathological phenotype is identified for the MSI-H tumors, which comprise approximately 15% of colorectal cancers, whereas MSI-L and MSS tumors appear to be phenotypically similar. MSI-H colorectal tumors are found predominantly in the proximal colon, have unique histopathological features, and are associated with a less aggressive clinical course than are stage-matched MSI-L or MSS tumors. Preclinical models suggest the possibility that these tumors may be resistant to the cytotoxicity induced by certain chemotherapeutic agents. The implications for MSI-L are not yet clear. (d) MSI can be measured in fresh or fixed tumor specimens equally well; microdissection of pathological specimens is recommended to enrich for neoplastic tissue; and normal tissue is required to document the presence of MSI. (e) The "Bethesda guidelines," which were developed in 1996 to assist in the selection of tumors for microsatellite analysis, are endorsed. (f) The spectrum of microsatellite alterations in noncolonic tumors was reviewed, and it was concluded that the above recommendations apply only to colorectal neoplasms. (g) A research agenda was recommended.

Colorectal Neoplasms↗
PubMed1998

Effects of piracetam on the performance of rats in a delayed match-to-position task.

1. The influence of acute and chronic treatment with piracetam on spatial working memory of rats was examined. A new version of an operant chamber "delayed match-to-position task" was used, in which the animals had to visit one randomly baited box out of three boxes ("choice boxes") in a front panel. Hereafter a delay period began, in which the subjects had to visit an alcove in the back panel ("reference box"). At the end of the delay the animals should return to the front panel and choose the same choice box that was baited before the delay, thereby obtaining a reward. 2. Rats were trained to a stable level of performance, measured as per cent correct responses during sessions of 20 trials. Additionally, the time spent between leaving the choice box and entering the reference box was recorded. Results were obtained from a single group of rats tested repeatedly under different experimental conditions. 3. Injections of scopolamine (0.6 mg/kg) significantly reduced the percentage of correct choices and increased the time spent to reach the reference box. The impairment of correct choices was reversed after chronic treatment with piracetam (250 mg/kg). However, the same treatment did not reverse the effect of scopolamine on the time performance. 4. Scopolamine also induced an increase of repetitive errors (a measure of perseverance), and the chronic treatment with piracetam caused full reversal of this increase. These results represent the first observation of a piracetam induced reversal of scopolamine impairments in a working memory test. 5. In normal animals not treated with scopolamine, acute injection of piracetam had no effect compared to saline injected controls, but chronic treatment with the nootropic significantly enhanced working memory performance. The drug did not affect the time used to reach the reference box.

Animals↗
PubMed2003

A panel of repeat markers for detection of microsatellite instability in murine tumors.

A substantial fraction of human malignancies lack functional DNA mismatch repair (MMR), accumulate mutations at high frequency, and exhibit microsatellite instability (MSI). In order to distinguish between MMR-competent and MMR-deficient malignancies, a consensus panel of microsatellite repeats has been adopted for MSI analysis of human tumors. There is no reference panel of repeats for MSI typing of murine malignancies. In this study, we present six new microsatellite repeat markers for MSI typing of mouse tumors. Analysis of polymerase chain reaction (PCR)-amplified tumor DNA from MMR-deficient and -proficient mice on denaturing polyacrylamide gels revealed that the new panel of microsatellites was more sensitive in detecting MSI than six commonly used CA(n) repeats. Using the new set of microsatellite markers, we demonstrated the presence of MSI in endometrial carcinoma and cancer precursors from diethylstilbestrol (DES)-treated mice, pointing to a possible role for loss of MMR in hormonally promoted endometrial tumorigenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Lymphocyte-predominant Hodgkin's disease. An immunohistochemical analysis of 208 reviewed Hodgkin's disease cases from the German Hodgkin Study Group.

There is wide consensus that lymphocyte predominance Hodgkin's disease (LPHD) represents a distinct clinicopathological entity of B-cell origin. However, inconsistent results of immunophenotyping studies and low confirmation rates among multi-center trials pose the question of whether LPHD really expresses heterogeneous marker profiles or whether it represents a mixture of morphologically similar entities. Among 2,836 cases reviewed by the German Hodgkin Study Group, immunophenotyping was performed on 1) cases classified or confirmed as LPHD by the reference panel (n = 104) or 2) cases not confirmed as LPHD but classified as classical HD (cHD) within the reference study trial (n = 104). In most cases, immunohistochemistry revealed a phenotype either LPHD-like (CD20+, CD15-, CD30-, CD45+) or cHD-like (CD15+, CD30+, CD20-, CD45-). In 27 cases, the immunophenotype was not fully conclusive. Additional markers for Epstein-Barr virus and CD57 and in situ hybridization for mRNA light chains allowed for a more clear-cut distinction between LPHD and cHD. However, in 25 of 104 cases, immunohistochemistry disproved the morphological diagnosis of LPHD of the panel experts, whereas 13 cases originally not confirmed as LPHD showed a LPHD-like immunopattern. Immunohistochemically confirmed LPHD cases showed a significantly better freedom from treatment failure (P = 0.033) than cHD; this was not observed in the original study classification based only on morphology (P > 0.05). Significantly better survival for LPHD cases improved from P = 0.047 (original study classification) to P = 0.0071 when classified by immunohistochemistry. Our results show that LPHD is a more immunohistochemical rather than a purely morphological diagnosis. Immunophenotyping of HD biopsies suspected of being LPHD is mandatory when a modified therapy protocol, that is, one different from those used in cHD, is discussed.

Adult↗