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

Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the β chain

Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ β chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence.analysis by flow cytofluorometry showed specific surface immunofluorescence in 1:100-1:1000 dilutions in lymphoblastoid and blood mononucleated cells. In the latter the binding was primarily confined to monocytes and a subpopulation of lymphocytes. It is concluded that locus-specific immunological reagents to distinguish between β chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide. © 1985

Determination of Total Homocysteine in Human Plasma by Isocratic High-Performance Liquid Chromatography

Peer Reviewe

Quantitation of serum free light chains does not compensate for serum immunofixation only when screening for monoclonal gammopathies

Background: Detection of plasma cell dyscrasias (PCD) requires screening of serum and urine for monoclonal proteins. Several studies have demonstrated increased sensitivity and specificity when measurement of serum free light chain (SFLC) is part of the screening protocol. In addition, omission of immunofixation (IFE) in the standard work-up that includes SFLC assay has been proposed. This study attempts to define the role of the SFLC assay in a screening strategy limited to serum only. It compares outcomes to a serum-only screening strategy that omits serum IFE. Methods: Serum from 691 patients was analysed for the presence of monoclonal protein using standard serum IFE, serum protein electrophoresis (SPE) and measurement of SFLC. Data were analysed retrospectively. Results: Specificity and sensitivity of abnormal SFLC-ratios for the detection of monoclonal protein using IFE were 96% and 41%, respectively. Eighteen patients with negative monospecific and Bence Jones IFE, but abnormal SFLC-ratios were identified. In most cases, this could be attributed to kidney and inflammatory disease or haematological disorders. In four cases, this resulted in further diagnostic investigation and light chain disease was later detected in two cases. Light chain disease was confirmed in one case but not confirmed in the other patient. In 14 patients, Bence Jones IFE was negative, although the concentrations of SFLC suggested the presence of monoclonal Bence Jones protein at concentrations detectable by IFE. Thus, either the anti-serum failed at detection, there was polymerisation of the free light chains or the SFLC assay overestimated protein concentrations. Simulating a work-up that included IFE only if abnormalities were detected by SPE or the SFLC assay would have resulted in 26% fewer IFEs being performed, but three patients with monoclonal proteins by IFE would have been missed. Conclusions: Abnormal SFLC concentrations are neither sensitive nor specific for the detection of monoclonal proteins by IFE. Not all PCD are accompanied by excessive production of SFLC, and several other conditions, such as renal disease are associated with increased SFLC concentrations. An abnormal SFLC-ratio is a specific marker for PCD, and occurs primarily in patients with haematological disease. If renal and inflammatory diseases are excluded, this should prompt further diagnostic investigation. Screening of serum without performing an IFE as a standard procedure leads to a reduction of sensitivity when compared to screening of serum that includes IFE. Clin Chem Lab Med 2009;47:1109–15.Peer Reviewe

No influence of 5-HTTLPR gene polymorphism on migraine symptomatology, comorbid depression, and chronification

Background. - The serotonergic system is thought to play an important role for mediating susceptibility to migraine and depression, which is frequently found comorbid in migraine. The functional polymorphism in the serotonin transporter gene linked polymorphic region (5-HTTLPR/SLC6A4) was previously associated with attack frequency and, thus, possibly with chronification. Objective. - We hypothesized that patients with the "s" allele have higher attack frequency and, paralleling results in depression research, higher scores of depression. Methods. - Genetic analysis of the SLC6A4 44 bp insertion/deletion polymorphism (5-HTTLPR) was performed in 293 patients with migraine with and without aura. Self-rating questionnaires were used for assessment of depression. Results. - Multinomial logistic regression analysis found no evidence for association of the 5-HTTLPR polymorphism with either depression or migraine attack frequency. Conclusion. - We were not able to demonstrate any influence of the serotonin transporter 5-HTTLPR polymorphism on migraine phenomenology (attack frequency or comorbid depression), thereby excluding this variant to be a common genetic denominator for chronic migraine and depression

Gene for integrin-associated protein (IAP, CD47): physical mapping, genomic structure, and expression studies in skeletal muscle.

Integrin-associated protein (IAP) is a widely expressed membrane protein with multiple functions in immunological and neuronal processes. Having physically mapped the IAP gene into a BAC/PAC contig covering approximately 1 Mb on human chromosome 3q13.1-q13.2, we determined the genomic organization of the gene, established its expression in skeletal muscle, and identified a novel splice variant. Our expression studies demonstrate expression of integrin- associated protein in the t-tubular system and the euchromatin of skeletal muscle cells where its function thus far is not known

Predictive value of S-100β and neuron-specific enolase serum levels for adverse neurologic outcome after cardiac surgery

AbstractObjectives: The aim of this study was to evaluate the time course of S-100β and neuron-specific enolase serum levels after cardiac surgery and their clinical relevance in predicting postoperative adverse neurologic outcomes; the 2 proteins are only released in peripheral blood in association with nervous system lesions. Methods: We neurologically assessed 190 consecutive patients undergoing elective cardiac operations for coronary artery bypass (n = 147), valve replacement (n = 29), or both (n = 14), before as well as after the operation. Postoperative outcome was classified as type I (uncomplicated), type II (confusion, agitation, disorientation, or epileptic seizures), or type III (stroke, stupor, or coma). Levels of S-100β and neuron-specific enolase were evaluated in venous blood samples drawn preoperatively and then daily in the first 5 postoperative days. Results: Levels of S-100β and neuron-specific enolase differed significantly among the 3 groups (type III > type II > type I) throughout the postoperative period and had a diagnostic specificity and specificity of 89% and 79%, respectively, in identifying patients with type III outcome. S-100β (but not neuron-specific enolase) levels were identified as significant independent predictors for type II and III outcomes (odds ratio 16.2, P < .0004). The same was true for duration of cardiopulmonary bypass (odds ratio 1.02, P < .006). Conclusions: Serum levels of S-100β are reliable markers for adverse neurologic outcomes after cardiac surgery. (J Thorac Cardiovasc Surg 2000;119:138-147