A polymorphic Alu insertion that mediates distinct disease-associated deletions.

Large deletions that are associated with insertions of Alu-derived sequence represent a rare, but potentially unique class of alterations. Whether they form by a one-step mechanism or by a primary insertion step followed by an independent secondary deletion step is not clear. We resolved two disease-associated SPAST deletions, which involve distinct exons by long range PCR. Alu-derived sequence was observed between the breakpoints in both cases. The intronic regions that represent the targets of potentially involved Alu retrotransposition events overlapped. Microsatellite- and SNP-based haplotyping indicated that both deletions originated on one and the same founder allele. Our data suggest that the deletions are best explained by two-step insertion-deletion scenarios for which a single Alu retrotransposition event represents the shared primary step. This Alu then mediated one of the deletions by non-homologous end joining and the other by non-allelic homologous recombination. Our findings thus strongly argue for temporal separation of insertion and deletion in Alu insertion-associated deletions. They also suggest that certain Alu integrations confer a general increase in local genomic instability, and that this explains why they are usually not detected during the probably short time that precedes the rearrangements they mediate.

Functional mutation analysis provides evidence for a role of REEP1 in lipid droplet biology.

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

ZNF804A and cortical structure in schizophrenia: in vivo and postmortem studies.

Recent evidence indicated that the ZNF804A (rs1344706) risk allele A is associated with better cognitive performance in patients with schizophrenia. Moreover, it has been demonstrated that ZNF804A may also be related to relatively intact gray matter volume in patients. To further explore these putatively protective effects, the impact of ZNF804A on cortical thickness and folding was examined in this study. To elucidate potential molecular mechanisms, an allelic-specific gene expression study was also carried out. Magnetic resonance imaging cortical thickness and folding were computed in 55 genotyped patients with schizophrenia and 40 healthy controls. Homozygous risk allele carriers (AA) were compared with AC/CC carriers. ZNF804A gene expression was analyzed in a prefrontal region using postmortem tissue from another cohort of 35 patients. In patients, AA carriers exhibited significantly thicker cortex in prefrontal and temporal regions and less disturbed superior temporal cortical folding, whereas the opposite effect was observed in controls, ie, AA carrier status was associated with thinner cortex and more severe altered cortical folding. Along with this, our expression analysis revealed that the risk allele is associated with lower prefrontal ZNF804A expression in patients, whereas the opposite effect in controls has been observed by prior analyses. In conclusion, our analyses provide convergent support for the hypothesis that the schizophrenia-associated ZNF804A variant mediates protective effects on cortex structure in patients. In particular, the allele-specific expression profile in patients might constitute a molecular mechanism for the observed protective influence of ZNF804A on cortical thickness and folding and potentially other intermediate phenotypes.

A spastic paraplegia mouse model reveals REEP1-dependent ER shaping.

Axonopathies are a group of clinically diverse disorders characterized by the progressive degeneration of the axons of specific neurons. In hereditary spastic paraplegia (HSP), the axons of cortical motor neurons degenerate and cause a spastic movement disorder. HSP is linked to mutations in several loci known collectively as the spastic paraplegia genes (SPGs). We identified a heterozygous receptor accessory protein 1 (REEP1) exon 2 deletion in a patient suffering from the autosomal dominantly inherited HSP variant SPG31. We generated the corresponding mouse model to study the underlying cellular pathology. Mice with heterozygous deletion of exon 2 in Reep1 displayed a gait disorder closely resembling SPG31 in humans. Homozygous exon 2 deletion resulted in the complete loss of REEP1 and a more severe phenotype with earlier onset. At the molecular level, we demonstrated that REEP1 is a neuron-specific, membrane-binding, and membrane curvature-inducing protein that resides in the ER. We further show that Reep1 expression was prominent in cortical motor neurons. In REEP1-deficient mice, these neurons showed reduced complexity of the peripheral ER upon ultrastructural analysis. Our study connects proper neuronal ER architecture to long-term axon survival.

Glutamate receptor δ 1 (GRID1) genetic variation and brain structure in schizophrenia.

Common genetic variation in the promoter region of the glutamate receptor delta 1 (GRID1) gene has recently been shown to confer increased risk for schizophrenia in several independent large samples. We analysed high-resolution magnetic resonance imaging (MRI) data from 62 patients with schizophrenia and 54 healthy controls using voxel-based morphometry (VBM) to assess the effect of single nucleotide polymorphism rs3814614 (located in the GRID1 promoter region), of which the T allele was identified as a risk factor in a previous association study. There were no effects of genotype or group × genotype interactions on total brain grey matter or white matter, but on regional grey matter. In healthy subjects, we identified a significant effect of rs3814614 genotype in the anterior thalamus (bilaterally), superior prefrontal cortex, and orbitofrontal cortex - in all cases with the homozygous risk genotype TT resulting in higher grey matter density. We did not find this association within the schizophrenia sample, where rs3814614 variation was only associated with grey matter reduction in TT homozygous subjects in medial parietal cortex and increased grey matter in right medial cerebellum. For white matter, we did not find significant genotype effects in healthy controls, and only minor effects within schizophrenia patients in the posterior temporal lobe white matter. Our data indicate that GRID1 rs3814614 genotype is related to grey matter variation in prefrontal and anterior thalamic brain areas in healthy subjects, but not in patients indicating a potential role of this schizophrenia candidate gene in thalamo-cortical functioning.

Mass spectometry-based protein patterns in the diagnosis of sepsis/systemic inflammatory response syndrome.

Early differential diagnosis of systemic inflammatory reactions in critically ill patients is essential for timely implementation of lifesaving therapies. Despite many efforts made, reliable biomarkers to discriminate between infectious and noninfectious causes of systemic inflammatory response syndrome (SIRS) are currently not available. Recent advances in mass spectrometry-based methods have raised hopes that identification of spectral patterns from serum/plasma samples can be instrumental in this context. We compared protein expression patterns from patients with SIRS of infectious and noninfectious origin. Plasma samples from 166 patients obtained under rigorously standardized preanalytical conditions were applied to Q10 and CM10 ProteinChips. Protein profiles were used to train and develop decision tree classification algorithms. Discriminatory peaks were isolated and identified. Classification trees distinguished patients with noninfectious SIRS with organ dysfunction following open heart surgery using cardiopulmonary bypass from those with severe sepsis or septic shock with distinct sensitivities and specificities. Results were validated in a blinded test set in two independent experiments and in a second independently collected test set. Discriminatory peaks at 13.8 and 55.7 kd were identified as transthyretin and α1-antitrypsin; the third protein at m/z 4,798 was assigned to a proteolytic fragment of α1-antitrypsin. Taken together, our data demonstrate that plasma protein profiling allows reproducible discrimination between patients with infectious and noninfectious SIRS with high sensitivity and specificity. However, rigorous standardization as well as considering drug-related interferences is essential when interpreting protein profiling studies. Identification of discriminatory proteins suggests a direct link between infectious-related protease activity and a sepsis-specific diagnostic pattern for discrimination of patients with SIRS.

Immediate diagnosis of ventriculits: evaluation of parameters independent of microbiological culture.

BACKGROUND: Generally accepted reference values in CSF diagnostics are not valid in cerebrospinal fluid (CSF) containing large amounts of blood. Residual blood may obscure ventriculitis as diagnostics largely depend on parameters such as cell count, lactic acid and total protein measurement. We sought to improve the diagnostics by evaluating a cytokine panel and soluble CD62L as markers of ventriculitis. In addition, we tested an algorithm of established parameters to predict ventriculitis in a specific patient collective. METHODS: Analysis was performed on ventricular CSF samples from 50 consecutive patients. Gram staining, microbiological culture, total cell count, total protein and CD62L expression on neutrophil granulocytes were analysed immediately. Cytokines and soluble CD62L were measured by flow cytometry. FINDINGS: Positive culture was detected in ten patients. Of all parameters tested only IL1-beta, IL8 and CD62L on neutrophils were significantly different between culture-positive and -negative patients. The highest predictive value was obtained when analysing IL1-beta. The predictive value of a parameter combination (IL6 in CSF, C-reactive protein and leukocytes in periphereal blood) was comparable to IL1-beta. Half of the patients in this analysis were identified as culture positive because of the lack of inflammatory response. CONCLUSIONS: IL1-beta and perhaps also IL8 provide very good analytical performance when looking for ventriculitis in patients with residual blood in CSF. Turn-around time is short, and results could be reported within 1 h for 24 h a day. In some patients application of glucocorticoids may result in restricted inflammatory response. Even in these patients IL1-beta provides a reliable parameter for the immediate diagnosis of ventriculitis.

Reduced cortical thickness is associated with the glutamatergic regulatory gene risk variant DAOA Arg30Lys in schizophrenia.

In light of current etiological concepts the glutamatergic system plays an essential role for the pathophysiology of the disorder, offering multiple options for new treatment strategies. The D-amino oxidase activator (DAOA) gene is closely connected to the glutamatergic system and its therapeutic and pathophysiological relevance for schizophrenia is therefore intensively debated. In a further step to shed light on the role of DAOA in schizophrenia, we aimed to investigate the association of the functional DAOA Arg30Lys (rs2391191) variant and cortical thickness in schizophrenia. Cortical thickness was computed by an automated surface-based technique (FreeSurfer) in 52 genotyped patients with schizophrenia and 42 healthy controls. Cortical thickness of the entire cortex was compared between risk carriers and non-risk carriers regarding the Arg30Lys polymorphism in patients and healthy controls on the basis of a node-by-node procedure and an automated clustering approach. Risk carriers with schizophrenia show significantly thinner cortex in two almost inversely arranged clusters on the left and right hemisphere comprising middle temporal, inferior parietal, and lateral occipital cortical areas. The clusters encompass an area of 1174 mm(2) (left) and 1156 mm(2) (right). No significant effect was observed in healthy controls.The finding of our study that the Arg30Lys risk variant is associated with a distinct cortical thinning provides new evidence for the pathophysiological impact of DAOA in schizophrenia. The affected areas are mostly confined to cortical regions with a crucial role in the ToM network and visual processing, which both can be influenced by glutamatergic modulation. Our finding thus underlines the importance of DAOA and related glutamatergic processes as a putative target for therapeutic interventions in schizophrenia.

CD62L on neutrophil granulocytes, a useful, complementary marker for the prediction of ventriculitis in blood-containing CSF.

OBJECTIVES: Presence of residual blood is a common problem in cerebrospinal fluid (CSF) diagnostics of ventriculitis. We hypothesised that neutrophil granulocytes in infected, blood-containing CSF lose CD62L expression. Therefore CD62L expression on neutrophils may present a complementary marker to distinguish between patients with residual blood and infection. DESIGNS AND METHODS: Evaluation was performed in 64 ventricular CSF samples sent to the laboratory for diagnostic investigation. Cell count, microbiological culture, total protein and flow cytometric analysis of CSF were performed. RESULTS: Cell counts and CD62L expression were significantly different between the culture positive and negative group. ROC-analysis revealed a significant predictive value for cell count and CD62L expression. Optimal cut-offs were calculated and a decision tree was established to predict a positive culture. CONCLUSIONS: Cell count and CD62L expression were predictive for a positive culture and the combination helped to increase specificity and sensitivity for the detection of ventriculitis in blood-containing CSF.

[Uncontrolled clinical study of the efficacy of ambulant fasting in patients with osteoarthritis]. / Unkontrollierte klinische Studie zur Wirksamkeit ambulanten Heilfastens bei Patienten mit Arthrose.

OBJECTIVE: To study the efficacy of fasting therapy according to Buchinger on pain, state of health, and articular function in patients with osteoarthritis. PATIENTS AND METHODS: Uncontrolled pilot study in which 30 patients (22 women, 8 men) with osteoarthritis (Kellgren stages I-III) of the hand (N = 10), hip (N = 8) and knee (N = 12) underwent ambulant fasting therapy according to Buchinger for 2 weeks with 3 pre-fast days, 8 fast days (300 kcal) and 4 re-feed days as well as follow-up 4 and 12 weeks afterwards. ASSESSMENT CRITERIA: Global intensity of pain (visual analogue scale, VAS); joint pain with activity, with start of walking, at rest (VAS); pressure pain threshold; articular function; health-related quality of life (SF-36 including Physical Component Score and Mental Component Score); Western Ontario and McMasters Universities Arthrose Index (WOMAC); painDETECT-questionnaire (Pfizer); analgesics; weight; body mass index (BMI); waist circumference; blood pressure; pulse and a variety of serological parameters. RESULTS: Pain, state of health, and articular function improved significantly; significant reduction in weight, BMI, and waist circumference during fasting and over the complete course of the study; analgesics could be reduced. No abnormalities in autonomous, metabolic, or blood parameters were observed. CONCLUSION: Medically supervised fasting can have a positive impact on the symptoms of patients with moderate osteoarthritis. This finding must be consolidated by controlled studies that include higher numbers of patients.

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.

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

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.

Comparison of six D-dimer assays for the detection of clinically suspected deep venous thrombosis of the lower extremities.

Deep venous thrombosis is a common disease that may lead to life-threatening embolism of the lung as a common complication. Therefore, early diagnosis followed by sufficient treatment is necessary to decrease mortality of this disease. D-dimer testing is established as a standard to rule out deep venous thrombosis in selected patient groups. However, there is no standardization among D-dimer assays, and a periodical comparison of assay performance in a select patient group is indispensable. We evaluated six commonly used D-dimer assays for their assay performance in an outpatient cohort with clinically suspected deep venous thrombosis. Although area under the curve for these assays did not differ significantly (0.83-0.88), differences in sensitivity (90-100%) and specificity (10-40%) of the assays were detected. Alternative cut-offs were established, and these cut-offs could enhance assay performance in some cases. This points to the fact that the manufacturers should more regularly review studies on the performance of their respective assays to widen the data basis for their recommended cut-offs and increase assay performance.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics.

The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical standardization and quality-control measures required for effective implementation into routine laboratory testing in order to generate clinically useful information. With new disease biomarker candidates, it will be crucial to design and perform clinical studies that can identify novel diagnostic strategies based on these techniques, and to validate their impact on clinical decision-making.

Direct analysis of clinical relevant single bacterial cells from cerebrospinal fluid during bacterial meningitis by means of micro-Raman spectroscopy.

Bacterial meningitis is a relevant public health concern. Despite the availability of modern treatment strategies it is still a life-threatening disease that causes significant morbidity and mortality. Therefore, an initial treatment approach plays an important role. For in-time identification of specific bacterial pathogens of the cerebrospinal fluid (CSF) and emerged antimicrobial and adjunctive treatment, microbiological examination is of major importance. This contribution spotlights the potential of micro-Raman spectroscopy as a biomedical assay for direct analysis of bacteria in cerebrospinal fluid of patients with bacterial meningitis. The influence of miscellaneous artificial environments on several bacterial species present during bacterial meningitis was studied by means of Raman spectroscopy. The application of chemometric data interpretation via hierarchical cluster analysis (HCA) allows for the differentiation of in vitro cultured bacterial cells and can also be achieved on a single cell level. Moreover as proof of principle the investigation of a CSF sample obtained from a patient with meningococcal meningitis showed that the cerebrospinal fluid matrix does not mask the Raman spectrum of a bacterial cell notably since via chemometric analysis with HCA an identification of N. meningitidis cells from patients with bacterial meningitis could be achieved.

Workflow restrictions on hematology analyzers XE-2100 and XS-800 when assaying pediatric samples with limited volume.

BACKGROUND: Samples with limited volume is a common problem in laboratories receiving samples from pediatric patients. Also, pediatric samples may contain nucleated red blood cells (NRBC) which distorts the white blood cell (WBC) count and which can only be measured by some automated cell counting systems. Differential counts are sometimes required, posing the question of validity of flagging depending on age of the patients and on predilutions. METHODS: We evaluated the hematology analyzers XE-2100 and XS-800 for their suitability in measuring hematological parameters in such samples. RESULTS: With the exception of the MCHC and partly the MCH, we observed very good agreement between complete blood counts (CBC) in diluted and undiluted samples. Dilution did not impair sensitivity in the clinically relevant range nor, accuracy of the NRBC count on XE-2100. Flagging was ineffective in undiluted samples from children<1 year of age and in all diluted samples when measuring differential counts. CONCLUSIONS: In summary, while automated measurement of CBC and NRBC is possible in diluted samples, measurement of differential counts is restricted by loss of flagging efficiency. In addition, flagging is also ineffective in children<1 year of age using the analyzers evaluated and should, for diagnostic purposes, be performed manually.

Evaluation of two different albumin depletion strategies for improved analysis of human CSF by SELDI-TOF-MS.

OBJECTIVES: Two different depletion strategies for removing albumin from human cerebrospinal fluid (CSF), Microcon Centrifugal Filter vs. Montage Albumin Deplete kit, were evaluated for improving protein profiling pattern and reproducibility in SELDI analysis. DESIGN AND METHODS: Pooled CSF was divided into 20 aliquots and these aliquots were subjected to SELDI analysis either after albumin depletion or without preprocessing. Protein profiles were obtained by using CM10, Q10 and IMAC chips. RESULTS: Both strategies resulted in reliable albumin depletion (<6.2 mg/L, filter; 8.1 mg/L, depletion kit). Investigated methods of albumin depletion showed no significant differences in coefficients of variation (CVs) of peak intensities compared to unprocessed CSF on almost all chip types. Peak intensities were significantly higher after albumin depletion compared to CSF without preprocessing on Q10 and on CM10 chips. Nevertheless, the two strategies of albumin depletion led to a decrease in the number of detected peaks on all chip types compared to unprocessed CSF, but several additional peaks, not detected in unprocessed CSF, occurred. CONCLUSIONS: This study demonstrates that reduction of sample complexity by albumin depletion of CSF can be performed without CV impairment. However, the significance of this strategy needs to be evaluated separately for each individual biomarker discovery study.

Evaluation of the XE-5000 for the automated analysis of blood cells in cerebrospinal fluid.

OBJECTIVES: Counting of cells in cerebrospinal fluid is an important clinical laboratory test and elevated white blood cell counts in cerebrospinal fluid are frequently seen in CNS disorders. Quantification of red blood cell concentrations in CSF may help to interpret certain diagnostic constellations and may result from subarachnoid haemorrhage, surgical procedures or contamination due to traumatic puncture. Table top analyser XE-5000 (Sysmex, Norderstedt, Germany) offers, beside its use as a haematology analyser, a protocol for the quantification of red and white blood cells in body fluids such as CSF including the differentiation between polymorphonuclear and mononuclear cells. A detection limit of 1 cell/mm(3) would render this device suitable for automated CSF analysis. DESIGN AND METHODS: White blood cell counting was compared between Fuchs-Rosenthal counting chamber and XE-5000 in 273 routinely collected lumbar and ventricular CSF samples. Red blood cell counting was compared between UF-100 and XE-5000. Differentiation was performed on a slide stained after Pappenheim and compared to the differential count of the XE-5000. RESULTS: Linearity was established between 1 and 10,000 cells/mm(3) for white blood cells and between 1000 and 110(3) particles/mm(3) for red blood cells. Functional sensitivity was established at 20 cells/mm(3) for white blood cell counting and at 1000 particles/mm(3) (lowest reported concentration) for red blood cell counting. When comparing between microscopic and automatic white blood cell counts no statistically significant slope and offset were detected in lumbar CSF samples while a significant slope and offset were detected when comparing ventricular CSF samples. Most patients were classified correctly according to their WBC count (non-pathologic, mildly, moderately, and highly elevated) by both methods although more patients had pathologic white blood cell counts on XE-5000. A significant slope and offset were detected when comparing red blood cell counts between UF-100 and XE-5000. CONCLUSIONS: In summary despite its high imprecision at low white blood cell counts (<20 particles/mm(3)) most patients were classified correctly and therefore XE-5000 is suitable for automated quantification of white blood cells in cerebrospinal fluid in a defined diagnostic setting. This could significantly improve automation in the relatively time- and manual work-intensive field of cerebrospinal fluid diagnostics. However, careful review of plausibility of the results continues to be compulsory.

Analysis of CYP7B1 in non-consanguineous cases of hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a neurodegenerative condition defined clinically by lower limb spasticity and weakness. Homozygous mutations in CYP7B1 have been identified in several consanguineous families that represented HSP type 5 (SPG5), one of the many genetic forms of the disease. We used direct sequencing and multiplex ligation-dependent probe amplification to screen for CYP7B1 alterations in apparently sporadic HSP patients (n = 12) as well as index patients from non-consanguineous families with recessive (n = 8) and dominant (n = 8) transmission of HSP. One sporadic patient showing HSP as well as optic atrophy carried a homozygous nonsense mutation. Compound heterozygosity was observed in a recessive family with a clinically pure phenotype. A heterozygous missense change segregated in a small dominant family. We also found a significant association of a known coding polymorphism with cerebellar signs complicating a primary HSP phenotype. Our findings suggest CYP7B1 alterations to represent a rather frequent cause of HSP that should be considered in patients with various clinical presentations.