Experiences with external quality assessment (EQA) in molecular diagnostics in clinical laboratories in Germany. Working Group of the German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM).

The German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM) have established an official working group on molecular diagnostics in the field of laboratory medicine. The group's objectives are to support the establishment of molecular biology methods for the use in diagnostics in German clinical laboratories. Towards this end, we have defined specific aims, which are 1) the implementation and extension of external quality assessment (EQA) schemes and methodological exercises offered to clinical diagnostic laboratories, 2) the establishment of a proficiency network and data base within the societies, 3) the implementation of an educational program in molecular diagnostic procedures for clinical chemists and laboratory physicians through the organisation of symposia and workshops and 4) the cooperation with other DGKC/DGLM working groups on shared aspects of laboratory analysis, e.

Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.

The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14).

A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.

Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro.

Several reverse transcriptase polymerase chain reaction (RT-PCR) assays have been described for the detection of circulating tumour cells in blood and bone marrow. Target mRNA sequences for this purpose are the cytokeratins (CK) 19 and 20, the carcinoembryonic antigen (CEA), and the prostate-specific antigen messages. In this study, we investigated biological factors influencing the specificity of the CK19 and CEA RT-PCR assays.

Kinetic and affinity constants of epitope specific anti-carcinoembryonic antigen (CEA) monoclonal antibodies for CEA and engineered CEA domain constructs.

Background: Carcinoembryonic antigen (CEA) is a human tumor antigen with the domain structure N-A1-B1-A2-B2-A3-B3, in which each domain is predicted to have an Ig-like fold and is known to bind epitope specific anti-CEA antibodies.

Objective: To determine the affinity constants of several domain specific anti-CEA antibodies using purified recombinant or synthetic domains.

Results And Conclusion: We have determined the kinetic and affinity constants of several anti-CEA antibodies for CEA, CEA domains (A3-B3) expressed in HeLa cells, and a synthetic peptide corresponding to the A3 domain using a BIAcore biosensor.

External quality assessment of molecular biology-based methods used in laboratories of clinical chemistry and human genetics.

The Reference Institute of Bioanalysis of the German Society of Clinical Chemistry has performed the first external assessment of molecular genetics methods used in medical diagnosis. The following procedures were tested: (I) DNA preparation from whole blood, (II) PCR amplification using "standard" primers, and (III) submarine agarose gel electrophoresis. Out of 50 participants, 45 returned samples for evaluation.

Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.

The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end.

Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA.

Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity.

Clinical evaluation of indium-111-labeled chimeric anti-CEA monoclonal antibody.

Unlabelled: Chimeric T84.66 (cT84.66) is a high-affinity (1.

Dysregulation of carcinoembryonic antigen group members CGM2, CD66a (biliary glycoprotein), and nonspecific cross-reacting antigen in colorectal carcinomas. Comparative analysis by northern blot and in situ hybridization.

Genes coding for CD66a (biliary glycoprotein), carcinoembryonic antigen (CEA) group member 2 (CGM2), and nonspecific cross-reacting antigen (NCA) are members of the human CEA gene subgroup. We investigated a series of 11 colorectal carcinomas by Northern blot and isotopic in situ hybridization (ISH), demonstrating underexpression of CD66a and CGM2 in the majority of the carcinomas as compared with the normal mucosa, whereas NCA was overexpressed. ISH for CD66a and CGM2 mRNA revealed that large areas of the carcinomas remained without or with only faint hybridization signals.

Expression of CD66a (human C-CAM) and other members of the carcinoembryonic antigen gene family of adhesion molecules in human colorectal adenomas.

Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis.

Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction.

The sensitive and specific detection of micrometastasis holds great promise for earlier staging of cancer patients. By amplification of tissue-specific gene expression, the reverse transcriptase polymerase chain reaction (rtPCR) readily detects single tumour cells in different tissues. An increasing number of rtPCR assays with possible relevance for routine laboratory diagnostic procedures is currently being reported in the literature.

Expression of a glycoprotein of the carcinoembryonic antigen family in normal and neoplastic sebaceous glands. Limited role of carcinoembryonic antigen as a sweat gland marker.

Background: Carcinoembryonic antigen (CEA) is a well-known marker for sweat gland differentiation in adnexal neoplasms.

Objective: The aim of this study was to examine the expression of glycoproteins of the CEA family, that is, CEA-180, nonspecific cross-reacting antigens (NCAs), and biliary glycoprotein (BGP), in sebaceous glands and in neoplasms with sebaceous differentiation.

Methods: Normal adult and fetal skin, hyperplasias, hamartomas, and neoplasms with sebaceous or follicular differentiation were stained immunohistochemically with a panel of polyclonal and monoclonal antibodies highly specific for CEA-180, NCAs, and BGP.

Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers.

Background: Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo.

Objectives: To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model.

Neoplasms with sweat gland differentiation express various glycoproteins of the carcinoembryonic antigen (CEA) family.

Carcinoembryonic antigen (CEA) is a well-established marker for sweat gland differentiation in adnexal neoplasms. In contrast to previous assumptions, CEA does not represent a single oncofetal antigen but comprises a family of homologous glycoproteins, i.e.

B-cell chronic lymphocytic leukemia associated with high serum IGE levels and pruriginous skin lesions: successful therapy with IFN-alpha 2b after failure on IFN-gamma.

Background: Chronic lymphocytic leukemia of the B-cell type (B-CLL) associated with highly elevated serum IgE levels and skin involvement has rarely been observed. Furthermore, not much is known about therapeutic strategies in such diseases.

Objective: We describe a 56-year-old male patient with a 5-year history of chronic and relapsing pruritic skin lesions as well as recurrent Staphylococcus aureus infections of the skin.

Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin.

The carcinoembryonic antigen (CEA) family comprises a group of glycoproteins including the classical CEA, nonspecific cross-reacting antigens (NCA), and biliary glycoprotein (BGP). CEA glycoproteins have been identified in many glandular and mucosal tissues. In view of their putative role in cell adhesion, protein sorting, and signal transduction, CEA glycoproteins are thought to be involved in embryogenesis, architectual integrity, and secretory mechanisms of glandular epithelia.

CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.

CD66a, also called biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and of the immunoglobulin superfamily. CD66a is the human homologue of Cell-CAM, a well-defined cell adhesion molecule of the rat. In the present study a monoclonal antibody specific for CD66a was used to locate CD66a in human tissues.

Initial experience evaluating 90yttrium-radiolabeled anti-carcinoembryonic antigen chimeric T84.66 in a phase I radioimmunotherapy trial.

Chimeric T84.66 (cT84.66) is a high-affinity (5 x 10(10) M-1) anti-carcino-embryonic antigen (CEA) IgG1.

Association of pp60c-src with biliary glycoprotein (CD66a), an adhesion molecule of the carcinoembryonic antigen family downregulated in colorectal carcinomas.

CD66a, also known as 'biliary glycoprotein (BGP)', is the human homologue of a cell adhesion molecule (CAM) of the rat (Cell-CAM). CD66a, which belongs to the carcinoembryonic antigen family and the immunoglobulin superfamily, is expressed in cells of myeloid and epithelial origin. The cytoplasmic domain of the major isoform of CD66a (CD66acyt) contains two tyrosine residues in amino acid motifs potentially interacting with protein tyrosine kinases of the Src family.

Diagnosis of micrometastases by the amplification of tissue-specific genes.

Micrometastases of solid tumors are most commonly detected by immunocytochemistry using monoclonal antibodies directed against tissue-specific gene products like cytokeratin-18 (CK-18) and the carcinoembryonic antigen (CEA). While CK-18 is a marker for epithelia in general, CEA is mainly employed in the detection of gastrointestinal and breast carcinomas. To improve the sensitivity and specificity of micrometastasis detection, we planned to establish polymerase chain reaction (PCR) assays for both markers.

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization.

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium.

Tumour diagnosis by PCR-based detection of tumour cells.

Tumour cells shed from solid primary tumours can be detected by the polymerase chain reaction (PCR) based on the selective amplification of mutated tumour genes or of genes expressed in a tissue specific manner. When tumour specific alterations are amplified, few tumour cells can be detected in excess of normal cells derived from the same tissue. Thus, malignant cells can be detected specifically in pancreatic juice, stool, urine, and sputum.