Prevention of the decrease in sensitivity of an amperometric glucose sensor in undiluted human serum.
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Blood Glucose↗
Biomedical subjects
B Linke
Publications and source records attributed to B Linke.
Prevention of the decrease in sensitivity of an amperometric glucose sensor in undiluted human serum
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Journal Article↗
Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas.
Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.
Base Sequence↗
Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.
The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.
Alleles↗
Automated molecular genetic DNA analysis for detecting B-cell non-Hodgkin's lymphoma in cytologic specimens.
OBJECTIVE: In fine needle aspiration cytology, conventional morphologic and immunocytologic criteria are often insufficient for distinguishing the mixed type of follicular lymphoma (NHL) from reactive lymphoid hyperplasia (RLH). For improving cytodiagnosis, we adapted computer-assisted polymerase chain reaction (PCR) assays on cytologic specimens. These novel techniques aim at fluorescence-based sequencing and sizing of PCR products that result from NHL-specific markers, such as the chromosomal t(14;18) (q32;q21) translocation and the CDR3 regions of expanded B-cell neoplasia. STUDY DESIGN: Aspirates from eight cases with mixed-type follicular NHL and from nine cases with a cytologic finding of RLH were investigated by PCR and with an automated laser scanning system. RESULTS: PCR detected the t(14;18) translocation in 5/8 B cell NHL samples and 1/9 RLH samples. By means of automated DNA sequencing, two of the five positive probes in the group of NHLs were identified as false positive. The CDR3 regions of expanded B-cell clones were found in four of eight NHL aspirates by means of PCR and computer-assisted analysis. CONCLUSION: This study proved the reliability of automated genetic assays for diagnosing low grade B-cell NHLs in aspirates since false positive results are excluded.
Biopsy, Needle↗
Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis.
Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific markers are increasingly being used in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) patients instead of the time consuming and labor intensive Southern analysis. In previous reports, no single common V beta and J beta sequence had been identified that allowed reliable amplification of the majority of rearranged T-cell antigen receptor (TCR)-beta V-D-J junctions at the DNA level because of the relatively large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes. In the present study we designed highly degenerate PCR primers directed against conserved sequences of the J beta genes. IN combination with a previously published consensus V beta primer, these J beta primers specifically amplify TCR- beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patients with T-cell lymphoproliferative disorders, one c-ALL patient, and five patients with nonmalignant diseases. T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls. Individual PCR products were sequenced after cloning. The nucleotide sequences of 96 randomly chosen recombinant vectors were determined. In the polyclonal controls all analyzed clones differed in their TCR-beta V-N(D)N-J junctions. In the T-cell lines, in all of the T-cell malignancies, and in the c-ALL, monoclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions. The PCR results were confirmed by automated fluorescence quantification and size determination of PCR products after separation in a high-resolution polyacrylamide gel. The procedure allows rapid and specific characterization of clonal TCR-beta rearrangements from genomic DNA and will significantly simplify current experimental approaches to identify and to quantitate malignant T cells during initial staging and follow-up of T-lineage NHL and ALL patients.
Automation↗
[Electron spin resonance spectroscopy investigations of fresh fruit. Evidence of treatment with ionizing radiation].
This paper describes the development of a method for the identification of irradiated fresh fruits by measurement of the e.p.r. spectra of pips, kernels or stones. Measurement parameters were optimized and the irradiation specific spectrum was assigned to a cellulose radical by comparison with the e.p.r. spectrum of pure cellulose. Several fruits especially different varieties of strawberries were examined giving the following results: Detectable minimum doses were between 0.4 kGy and 0.9 kGy and the intensity of the irradiation specific signals was found to be linear up to doses of 11 kGy. The lifetime of the specific radicals (at room temperature and at deep freezing temperatures) was long enough compared to the storage time of fresh fruits. Additional information about the nature of the unspecific central signal was gained measuring the samples which were stored at different temperatures. The main conclusion of this study is that the e.p.r. method seemes to be well suited for the use in routine control and should be tested in an intercomparison to establish a routine method for the identification of irradiated fresh fruits.
Dose-Response Relationship, Radiation↗
Localized gastric non-Hodgkin's lymphoma of high-grade malignancy in patients with pre-existing chronic lymphocytic leukemia or immunocytoma.
Analyses for clonality in cases of Richter's syndrome have provided evidence for a clonal evolution of high-grade lymphoma in most patients, while in others an independent cellular clone seems to exist in the secondary neoplasm. Richter's syndrome with an isolated high-grade lymphoma of the stomach has been rarely reported in patients with pre-existing B cell chronic lymphocytic leukemia (CLL). We investigated four cases of CLL or lymphoplasmacytoid immunocytoma (LPIC) with development of a localized high-grade B cell lymphoma in the stomach. Southern blotting showed different rearrangements of the immunoglobulin light and heavy chain genes in the tumor cells of the low-grade lymphoma and the gastric tumor in two cases. Comparison of the DNA sequences of the CDR3 region of the immunoglobulin genes revealed different clones in another case. By means of chromosomal in situ hybridization, trisomy 3 was detected in two cases of high-grade lymphoma of the stomach, but not in the cells of the associated low-grade tumor. Our findings indicate that high-grade non-Hodgkin's lymphomas arising localized in the stomach of patients with CLL or immunocytoma are not clonally related to the pre-existing low-grade lymphoma and, therefore indeed, present true secondary neoplasms.
Aged↗
Identification and structural analysis of rearranged immunoglobulin heavy chain genes in lymphomas and leukemias.
The third complementarity determining region (CDR3) of the hypervariable domain of immunoglobulin heavy chain (IgH) genes represents a highly variable and clone-specific IgH-CDR3 sequences in 10 non-Hodgkin's lymphomas (NHL), five chronic lymphocytic leukemias (CLL) and five acute lymphoblastic leukemias (ALL) of B cell lineage. The IgH-CDR3 sequences were amplified using DNA extracted from clinical specimens (bone marrow, peripheral blood and fresh-frozen or paraffin-embedded lymph nodes) by a semi-nested PCR with consensus primers directed to conserved regions within the variable (VH) and the joining (JH) gene segments. In 17/20 samples (85%), a distinct IgH-CDR3 PCR product was obtained. Individual PCR products were sequenced after cloning. The nucleotide sequences of 134 randomly chosen recombinant vectors were determined demonstrating in 17/20 cases (85%) monoclonal VH-N-DH-N-JH junctions. Analysis of PCR products by temperature-gradient gel electrophoresis (TGGE) confirmed the specificity of the IgH-CDR3 PCR/sequencing results. Moreover, the combination of PCR/TGGE technology allowed the rapid and specific characterization of clonal IgH-CDR3 junctions in B cell proliferations by direct sequencing even in the presence of admixed polyclonal B cells.
Base Sequence↗
Use of UITma DNA polymerase improves the PCR detection of rearranged immunoglobulin heavy chain CDR3 junctions.
The development of rapid PCR protocols for amplification of rearranged IgH gene sequences has greatly facilitated the identification of clonal IGH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias. However, the 15-35% incidence of false negative results with this approach has been a constant and unresolved problem. To assess the reliability of a previously published framework region 3 (FR3A) IgH-CDR3-PCR for detection of monoclonal IgH gene rearrangements we compared the PCR and Southern results in a series of 44 NHL and leukemias of B cell lineage showing a JH-rearrangement in Southern analysis with genomic DNA and hybridization with a IgH joining region (JH) probe. IgH-CDR3 regions were amplified using DNA extracted from clinical specimens by PCR using fluorescent dye-labeled consensus primers homologous to conserved regions within the variable (VH) and the joining (JH) gene segments. The PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. With commonly used DNA polymerases monoclonal IgH-CDR3 junctions were identified in 36/44 samples (82%). However, in the remaining eight cases (18%) with pathohistologically clearly demonstrated B cell malignancies which were also monoclonal on JH-Southern analysis, monoclonality could be demonstrated by FR3A-IgH-CDR3-PCR only with the proofreading UITma DNA polymerase. In four of these monoclonal VH--N--DH--N--JH junctions sequence analysis was performed which showed a point mutation in one and a single nucleotide deletion at the 3' terminus of the primer target site in the other case. In the remaining two cases no primer mismatches could be identified. Thus we conclude that the marked improvement of the PCR-detection rate of monoclonal IgH-CDR3 junctions was achieved at least in part due to the ability of UITma DNA polymerase to remove mismatched bases at the 3' terminus of the primers with respect to the target during the first amplification cycles. Our results suggest, that UITma is the DNA polymerase of choice for amplification of IgH-CDR3 junctions with consensus FR3A-VH- and JH-primers.
Base Sequence↗
Characterization of clone-specific rearrangement T-cell receptor gamma-chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing.
The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T-NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T-cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.
Base Sequence↗
[Electron spin resonance measurements on dried fruit. Carbohydrate composition and ESR signal structure of irradiated fruit].
While in a previous work the ESR spectroscopic detection of irradiated dried fruits was reported, in this paper liquid chromatographic determination of the carbohydrate fraction of these fruits is introduced and connected with the ESR results. After irradiation of dried fruits three different types of ESR spectra are observed. In most cases the dried fruits can be attached to these various types by means of their sugar composition. It was also found that the ESR spectra observed for sucrose-rich fruits are very similar to that of pure sucrose. The structure of the ESR spectra can change with storage. Probably, radical rearrangement reactions in the samples are responsible for these changes.
Carbohydrates↗
Amperometric biosensor for in vivo glucose sensing based on glucose oxidase immobilized in a redox hydrogel.
A potentially implantable glucose sensor, based on glucose oxidase immobilized in a redox hydrogel, is considered. The redox hydrogel consisted of glucose oxidase immobilized in a cross-linkable poly(vinylpyridine) complex of [Os(bis-bipyridine)2Cl]+1/+2 that communicates electrically with the flavin adenine dinucleotide (FADH2) redox centres of the glucose oxidase. The implantable electrode consisted of a Teflon insulated platinum wire (0.25 mm diameter) which was coated at the tip with a cross-linked redox polymer/glucose oxidase film and covered with a thin layer of polycarbonate. In a three-electrode system at +400 mV (Ag/AgCl) the response to increasing glucose concentrations in isotonic phosphate buffer and human plasma was approximately 0.2-0.3 nA/mM, linear in the range between 0 and 15 mM glucose. No oxygen dependence was observed. To determine the in vivo performance, the electrode was implanted into the subcutaneous tissue of a dog. The sensor currents after an oral glucose load paralleled the plasma glucose measurements, with a time lag of 10 min. Three-day implantations in cultured cells showed that the electrode did not affect the growth and differentiation of cell monolayers.
Animals↗
[Characterization of clonal B-cell populations in gastric MALT lymphomas and chronic gastritis by means of the polymerase chain reaction].
Amplification of the CDR3-region of the immunoglobulin (Ig) heavy chain gene rearrangement by means of the PCR yielded clonal products in the tumor DNA of 12 high or low grade gastric B-cell lymphomas of MALT-type. In four cases, additional clonal bands were found in different areas of tumor free mucosa diagnosed as chronic gastritis associated with Helicobacter pylori (HP). Most of these small clonal populations were found to share identical DNA sequences in their clone specific CDR3-regions with the main lymphoma in each patient; a finding consistent with the multifocal character of the disease. In two cases however, single clonal populations with different CDR3-regions revealed the existence of rare additional clonal B-cell-populations not related to the lymphoma and therefore possibly representing further independent foci.
B-Lymphocytes↗
[Identification of irradiated pasteurized egg products: a combined method for use in routine control].
Pasteurized egg products (whole egg, egg yolk and egg white) were tested for irradiation treatment in the German food control laboratories in Oldenburg/Niedersachsen and Kassel/Hessen as well as in the food irradiation laboratory of the German federal health office. Gas chromatographic/mass spectrometric measurements on the fat components of egg-products showed clearly whether the product had been irradiated or not. While in unirradiated samples no traces of special hydrocarbons (according to the fatty acid composition of egg) and no traces of the irradiation-specific compound 2-Dodecyl-cyclobutanone were found, irradiated control samples as well as products of two Belgian suppliers contained these substances. Additionally, regarding the rather high time consumption of gas chromatography, electron spin resonance (ESR)-measurements were carried out on the packaging material of egg products. Irradiated packaging material (cellulose) could be easily detected by the appearance of a signal pair in the ESR spectrum (cellulose radical). ESR measurements are very fast and easy to perform so that this method can be used for screening. Microbiological investigations showed remarkably reduced total numbers of microorganisms for some irradiated samples, but the microbiological status is influenced by other factors like storage-time and -temperature, so that microbiological tests can not be used successfully for screening on irradiation treatment.
Animals↗
The function of a hydrogen peroxide-detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma.
Electroenzymatic glucose sensors implanted into sub-cutaneous (s.c.) tissue of human subjects and experimental animals exhibit lower sensitivities to glucose than in buffer solutions before implantation. The mechanism of the decrease of sensitivity is not known. Sensors used in this study were fabricated from platinum wires (diameter 0.125 mm) with covalently bound glucose oxidase at the tip of the wire. After coating the tip with polyurethane, wires were placed into 27 gauge steel needles. Sensors were operated potentiostatically at 700 mV against Ag/AgCl pseudo-reference electrodes. These sensors were implanted s.c. in 6 diabetic patients for 7 h. In 4 patients, sensors were responsive to successive increases of plasma glucose levels. Mean sensitivity to glucose in s.c. tissue was 29% of in vitro sensitivity. In 2 patients there was a sudden decrease of sensor currents, unrelated to glucose, shortly after implantation. Sensors were inhibited in human plasma to a similar extent. When sensors were exposed to native plasma and to plasma ultrafiltrate (mol. wt. < 10 kDa) for 10 h, identical decreases of signals were found. Exposure to dialysed plasma (mol. wt. > 12 kDa) caused much less decrease of sensor signals. Losses of sensor sensitivities to glucose in s.c. tissue and in plasma were totally reversible upon re-exposure of sensors to buffer solutions. We conclude that sensor inactivation in plasma and possibly in s.c. tissue is caused by low molecular weight substances not retained by the polyurethane membrane.
Adult↗
[Electron spin resonance spectroscopy as probes for spices. Detection of treatment with ionizing radiation].
Detection of irradiated spices by electron spin resonance (ESR) measurements was not successful in the past because a central line of unknown origin was detected in the ESR-spectra of both irradiated and unirradiated samples. Identification of irradiated samples by measuring the increase of intensity of this signal after irradiation is limited because the signal intensity decreases over a period of some weeks of storage and reaches the range of unirradiated samples. By changing the measurement conditions (low microwave power) we could detect two additional lines on both sides of the main signal. This line pair appears only in the spectra of irradiated spices. A similar line pair was found in the spectra of irradiated nutshells and possibly derives from cellulose radicals in the sample. For some spices, especially paprika, the identification of irradiated samples by detecting these additional lines was possible even after relatively long periods of storage.
Condiments↗
[Status of the development of electron spin resonance measurement for the detection of irradiated food].
Electron spin resonance spectroscopy can be used for the detection of irradiation of various groups of foodstuffs. The results of ESR-measurements on irradiated meat and fish and fresh fruit, as well as dried fruit, spices and nuts as performed by the food irradiation laboratory of the German Federal Health Office are summarized in this report. For the detection of irradiated meat and fish, we examined the bones. Using the results from 10 different animal bones, we were able to develop an official method according to the German law section 35 LMBG. A similar routine method for fish will be established in 1992 (at the moment, an intercomparison with German food control laboratories is in progress). Irradiated dried fruit can be identified easily, because unirradiated samples give no ESR-spectra, while irradiated fruit show a partially resolved spectrum, which is caused by radiation induced sugar radicals. Interestingly, the structure of the resulting spectra is not identical for all irradiated species of fruit. We found three different types of ESR-spectra for irradiated dried fruit. Irradiated nutshells show an ESR-spectrum which reveals two additional lines (from cellulose-radicals) beside the main signal, while unirradiated samples show only the main signal. An official method for identifying irradiated nuts will be proposed in 1992. Irradiation specific ESR-signals of the cellulose radical were not only found for nutshells but also for fresh fruit and some spices, while most of the irradiated spices and herbs could not be identified by ESR-measurements.
Animals↗