Communication patterns in families affected by parental cancer from the healthy parents' perspective-process evaluation of the complex intervention Family-SCOUT.

PURPOSE: Within families affected by parental cancer, open communication impacts the well-being of parents and their children; however, limited research exists on communication patterns in these families. This sub-study addresses this through the Family-SCOUT study, a multicenter, prospective, interventional, and non-randomized investigation with intervention (IG) and control group (CG). The purpose of this sub-study was to identify and compare the differences in communication patterns between the IG and CG as part of the process evaluation. The research question was addressed in both groups: What communication patterns do healthy parents perceive within their families? METHODS: Using a qualitative approach, the study involved interviewing healthy parents as surrogates for their families. The interviews were audio-recorded, transcribed, and coded using a template analysis. The resulting data were analyzed at the group level. RESULTS: Twenty-three interviews were conducted in the IG and 27 interviews in the CG. The analysis of themes centered on communication patterns as seen in the family structure. Both groups exhibited instances of open communication about fears and wishes as well as the use of child-friendly language when discussing cancer. Notable differences were observed: challenges in open communication with children were sorely reported in CG interviews, and "the illness is discussed when necessary" was sorely described in IG interviews. CONCLUSION: This study underscores the need to address and encourage open communication within families with parental cancer.

[Gender differences in depression]. / Geschlechtsspezifische Aspekte bei depressiven Erkrankungen.

Depression is one of the most prevalent and debilitating diseases. In recent years there has been increased awareness of sex- and gender-specific issues in depression. This narrative review presents and discusses differences in prevalence, symptom profile, age at onset and course, comorbidity, biological and psychosocial factors, the impact of sexual stereotyping, help-seeking, emotion regulation and doctor-patient communication. Typically, women are diagnosed with depression twice as often as men, and their disease follows a more chronic course. Comorbid anxiety is more prevalent in women, whereas comorbid alcohol abuse is a major concern in men. Sucide rates for men are between three and five times higher compared with women. Although there are different symptom profiles in men and women, it is difficult to define a gender-specific symptom profile. Socially mediated gender roles have a significant impact on psychosocial factors associated with risk, sickness behavior and coping strategies. In general, too little attention has been paid to the definition and handling of depression and the gender-related requirements it makes on the healthcare system.

Effectiveness of a comprehensive support program for families with parental cancer (Family-SCOUT): results of a multicenter non-randomized controlled trial.

BACKGROUND: Cancer patients with minor children but also their families suffer from significant psychological distress and comorbidity. Protective factors predicting successful coping are well known. Corresponding systematic interventions are rare and limited by access barriers. We developed a comprehensive family-centered intervention for cancer patients with at least one dependent minor. PATIENTS AND METHODS: Family-SCOUT represents a multicentric, prospective, interventional, and controlled study for families with parental cancer and their minor children. In the intervention group (IG), all family members were addressed using a care and case management approach for nine months. Families in the control group (CG) received standard of care. Participating parents were asked to complete the Hospital-Anxiety-Depression-Scale (HADS) questionnaire at enrolment (T0) and after 9 months (T2). The primary outcome was a clinically relevant reduction of distress in at least one parent per family, measured as minimal important difference (MID) of ≥1.6 in the HADS total score. The percentage of families achieving MID is compared between the IG and CG by exact Fisher's test, followed by multivariate confounder analyses. RESULTS: T0-questionnaire of at least one parent was available for 424 of 472 participating families, T2-questionnaire after 9 months was available for 331 families (IG n = 175, CG n = 156). At baseline, both parents showed high levels of distress (HADS total: sick parents IG: 18.7 ± 8.1; CG: 16.0 ± 7.2; healthy partners: IG: 19.1 ± 7.9; CG: 15.2 ± 7.7). The intervention was associated with a significant reduction in parental distress in the IG (MID 70.4% in at least one parent) compared with the CG (MID 55.8%; P = 0.008). Adjustment for group differences from specific confounders retained significance (P = 0.047). Bias from other confounders cannot be excluded. CONCLUSIONS: Parental cancer leads to a high psychosocial burden in affected families. Significant distress reduction can be achieved through an optimized and structured care approach directed at the family level such as family-SCOUT.

[Physical and psychological violence perpetration and violent victimisation in the German adult population: results of the German Health Interview and Examination Survey for Adults (DEGS1)]. / Körperliche und psychische Gewalterfahrungen in der deutschen Erwachsenenbevölkerung: Ergebnisse der Studie zur Gesundheit von Erwachsenen in Deutschland (DEGS1).

Violence is of considerable relevance to Public Health. It was the aim of the violence screening implemented as part of the"German Health Interview and Examination Survey for Adults" (DEGS1) to assess data on physical and psychological violence in various social environments (partnership, family, workplace, public space). For the first time as part of a nationally representative health survey, the data was collected from the perspective of victim and perpetrator both among women and men. The study population was comprised of 5939 participants aged between 18 and 64 years. Approximately every 20th participant reported being the victim of physical violence in the preceding 12 months, men significantly more frequently than women. With regard to the frequency of being the perpetrator of physical violence (overall prevalence 3.7 %) there were no significant differences between the sexes. Psychological victimisation was reported by every fifth participant and overall perpetrating psychological violence was reported by every tenth. Women tended to be more frequent the victims but they were also significantly more frequently the perpetrators of both physical and psychological violence in the domestic area (partnership, family). In contrast, men more frequently report being both the perpetrator and the victim of violence in the workplace and in the public space. Young adults between 18 and 29 years as well as persons of low socioeconomic status were consistently more frequently affected by violence although there were exceptions with regard to psychological violent victimisation. More than three-quarters of the victims of physical violence reported being greatly or extremely affected in their well-being by the violence and in the case of psychological violence the rate was about approximately 60%. Overall, the traumatic experience as a consequence of experiencing physical and psychological violence was considerably higher, especially in the case of domestic violence (partnership, family). Overall, women reported a greater sense of wrongdoing following violence perpetration than men; as to the perpetration of violence towards a partner, however, there was no difference between the sexes in this regard. An English full-text version of this article is available at SpringerLink as supplemental.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome.

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Molecular discrimination of Hyalomma tick species serving as reservoirs and vectors for Crimean-Congo hemorrhagic fever virus in sub-Saharan Africa.

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies.

Imaging Mass Spectrometry and Proteome Analysis of Marek's Disease Virus-Induced Tumors.

The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors.IMPORTANCE Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.

Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance.

ATP-sensitive potassium (K(ATP)) channels are bifunctional multimers assembled by an ion conductor and a sulfonylurea receptor (SUR) ATPase. Sensitive to ATP/ADP, K(ATP) channels are vital metabolic sensors. However, channel regulation by competitive ATP/ADP binding would require oscillations in intracellular nucleotides incompatible with cell survival. We found that channel behavior is determined by the ATPase-driven engagement of SUR into discrete conformations. Capture of the SUR catalytic cycle in prehydrolytic states facilitated pore closure, while recruitment of posthydrolytic intermediates translated in pore opening. In the cell, channel openers stabilized posthydrolytic states promoting K(ATP) channel activation. Nucleotide exchange between intrinsic ATPase and ATP/ADP-scavenging systems defined the lifetimes of specific SUR conformations gating K(ATP) channels. Signal transduction through the catalytic module provides a paradigm for channel/enzyme operation and integrates membrane excitability with metabolic cascades.

High-level expression of the retinoic acid receptor beta gene in normal cells of the uterine cervix is regulated by the retinoic acid receptor alpha and is abnormally down-regulated in cervical carcinoma cells.

Retinoic acid (RA) is essential for regulation of epithelial cell differentiation. The intracellular effects of RA are mediated by RA-binding nuclear receptors, including the RA receptors (RARs) alpha, beta, and gamma. The ligand-activated receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. One target gene is the RAR beta gene, which encodes a potential tumor suppressor. Loss of RA inducibility of RAR beta gene expression is assumed to play a role in the development of several types of human carcinomas, including carcinomas of the uterine cervix. We have analyzed RAR beta gene expression in normal cervical cells and in cervical carcinoma cell lines. The results show that the RAR beta mRNA levels are high and RA inducible in the primary keratinocytes, whereas they are low and not inducible or only slightly inducible by RA in all of the cervical carcinoma cell lines analyzed. The basal and the RA-induced RAR beta mRNA levels tend to increase with senescence of the normal cells. Fusion of primary ectocervical keratinocytes with HeLa cervical carcinoma cells revealed that the characteristics of RAR beta gene expression of the normal cells are dominant over that of the tumor cells. Using synthetic retinoids with receptor-preferential agonist activities and a RAR alpha-specific antagonist, we show that RAR alpha is the major endogenous RAR subtype for induction of RA-dependent RAR beta gene expression. Taken together, our results indicate that abnormal downregulation of RAR beta gene expression may be an important step in the multifactorial process of cervical carcinogenesis.

Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane.

A simple and rapid method is described for the purification of two alphaherpesviruses, pseudorabies virus (PrV) and bovine herpesvirus 1, by chromatography on a cation exchange membrane. Cell culture supernatants were passed over a sulfonic-acid modified filter membrane and virions were eluted with a potassium chloride-containing buffer. Over 85% of the virus was eluted within a single fraction and specific infectivity of the resulting virus preparation was over 10-fold higher than that of sucrose gradient-purified virions. Cation exchange was also used for purification of PrV mutants deleted in several glycoproteins which grow in cell culture to titers 10- to 100-fold lower than those obtained by wildtype PrV. For PrV, the presence of non-essential glycoprotein gC, which mediates interaction of virions with cell surface heparin sulfate during attachment, was crucial for the successful purification by cation exchange.

Capillary electrophoretic separation of uncharged polymers using polyelectrolyte engines. Theoretical model.

We recently demonstrated that the molecular mass distribution of an uncharged polymer sample can be analyzed using free-solution capillary electrophoresis of DNA-polymer conjugates. In these conjugates, the DNA is providing the electromotive force while the uncharged polydisperse polymer chains of the sample retard the DNA engine with different amounts of hydrodynamic drag. Here we present a theoretical model of this new analytical method. We show that for the most favourable, diffusion-limited electrophoresis conditions, there is actually an optimal DNA size to achieve the separation of a given polymer sample. Moreover, we demonstrate that the effective friction coefficient of the polymer chains is related to the stiffness of the two polymers of the conjugate, thus offering a method to estimate the persistence length of the uncharged polymer through mobility measurements. Finally, we compare some of our predictions with available experimental results.

Separation of DNA sequencing fragments using an automated capillary electrophoresis instrument.

Rapid, high-resolution separation of DNA sequencing fragments by capillary gel electrophoresis using an automated, commercially available instrument is presented. The effect of column lengths and electric field strength on the resolution of sequencing fragments as well as the sensitivity of laser-induced fluorescence (LIF) detection was investigated. Using a short capillary of 20 cm length, which results in a U-shape of the capillary in the capillary cartridge, very high separation efficiency, up to 17 x 10(6) theoretical plates per m, is obtained. Analysis of the band broadening factors revealed that the resolution on the short column is predominantly determined by axial diffusion and to a minor extent by detection zone width. Presumably due to the coiling of longer capillaries in the capillary cartridge, increasing the capillary length does not increase the separation efficiency as predicted for diffusion-limited separation. The concentration limit of detection (signal-to-noise ratio = 2) is 0.2 x 10(-12) M of fluorescein-labeled oligonucleotide primer under the separating conditions for DNA sequencing samples. Increasing the electric field strength from 100 to 175 V/cm improved resolution and at the same time approximately doubled the sequencing speed. Fragments up to 500 nucleotides in length are resolved in less than 50 min.

Figuring Miniature Aspherics-lon Polishing.

A technique is described for accurately polishing extremely small areas by ion machining. A graphite mask is prepared with a pulsed laser cutting tool. Prepared under computer control, this mask defines the contour of the cavity to be formed in a glass tube. Two methods for measuring final contour of the tube are described.

[Assisted suicide in dementia. Ethical considerations between paternalism and autonomy]. / Sterbehilfe bei Demenz. Ethische Uberlegungen zwischen Paternalismus und Autonomie.

This article discusses the problem of euthanasia presenting the case of an 82-year-old man with progressive dementia. Difficulties encountered during daily clinical work are described and analysed, in order to clarify decisions on ethical, legal and professional medical grounds. General decisions concerning life-sustaining measures may be qualitatively improved if the situation of the individual is constantly assessed and considered within the treatment process.

Improvement of agitation and anxiety in demented patients after psychoeducative group intervention with their caregivers.

OBJECTIVES: It has been convincingly demonstrated that in dementia, psychoeducative group intervention with caregivers positively impacts on motivation for care and satisfaction of the caregivers. It has, however, been neglected to examine the effect of psychoeducative group intervention on the behavioural and psychological symptoms of the demented patients. METHODS: In a 3-month, expert-based and conceptualized group intervention with caregiving relatives of demented patients we investigated whether behavioural and psychological symptoms may improve and which of a set of independent variables may predict improvement. RESULTS: The 3-month group intervention yielded a significant improvement in agitation and anxiety of the demented patients. The presence of an additional somatic disease in the patients and male gender predicted a less positive outcome of the intervention related to the presence of agitation. CONCLUSIONS: This study demonstrated that psychoeducative group intervention with the caregivers of demented patients is helpful for the demented patients themselves. This evidence of a positive mediator effect of the group intervention on the behavioural and psychological symptoms of the patients underscores the importance of nonpharmacological strategies in the treatment of dementia.

Glycoproteins gIII and gp50 play dominant roles in the biphasic attachment of pseudorabies virus.

Virus infections are initiated by adsorption of virions to target cells. This step is mediated by viral attachment proteins that interact with specific cellular receptors. It has previously been shown that the alphaherpesvirus pseudorabies virus (PrV) attaches to cells by way of a process that involves the viral envelope glycoprotein gIII (gC) and a heparin-like cellular surface moiety (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat (1990) J. Virol. 64, 278-286). To gain further insight into adsorption of PrV different incubation protocols and an isogenic set of glycoprotein deletion mutants were analyzed. Here we show that attachment of wildtype PrV to target cells can be divided into three distinct stages: After primary adsorption, virus cannot be removed by a thorough wash with PBS but can be displaced by exogenous heparin defining a heparin-sensitive adsorption step. This primary adsorption at 0 degrees converts with a half-time of approximately 20 min into a heparin-resistant binding where attached virus is no longer sensitive to exogenous heparin. The presence of heparin during the adsorption process leads to a heparin-independent basal virus binding to cells. Analysis of null mutants in six PrV glycoproteins confirmed that the nonessential glycoprotein gIII (gC) is a major determinant of primary adsorption. The presence of both gIII and the essential glycoprotein gp50 (gD) was shown to be critical for heparin-resistant binding. The importance of gp50 for this process was demonstrated in two isogenic wildtype PrV and gp50- mutant pairs. Similar results were obtained with a mutant bovine herpesvirus 1 lacking the gD-homologous glycoprotein gIV, indicating a general role for the gD-homologous proteins in stable attachment of alphaherpesviruses. Since the gD-homologs are also involved in penetration they might link the processes of viral attachment and entry.

Identification of cell surface molecules that interact with pseudorabies virus.

The alphaherpesvirus pseudorabies virus (PrV) has been shown to attach to cells by interaction between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). A secondary binding step requires gD and presumably another, hitherto unidentified cellular receptor. By use of a virus overlay protein binding assay (VOPBA), cosedimentation analyses, and affinity chromatography, we identified three species of cell membrane constituents that bind PrV. By treatment with EDTA, peripheral HSPGs of very high apparent molecular mass (>200 kDa) could be extracted from Madin-Darby bovine kidney cells. Binding of PrV to these HSPGs in the VOPBA was sensitive to enzymatic digestion with heparinase or papain. Cosedimentation analyses indicated that binding between PrV and high-molecular-weight HSPG depended on the presence of gC in the virion. In addition, adsorption of radiolabeled PrV virions to cells could be inhibited by the addition of purified high-molecular-weight HSPG. By using urea extraction buffer, a second species of HSPG of approximately 140 kDa could be solubilized. Binding of PrV to this HSPG in the VOPBA was also dependent on the presence of heparan sulfate, since reactivity was abolished after suppression of glycosaminoglycan biosynthesis with NaClO3 and after heparinase treatment. In addition to HSPG, in cellular membrane extracts obtained by treatment with mild detergent, a 85-kDa membrane protein was demonstrated to bind PrV in the VOPBA and affinity chromatography. In summary, we identified three species of cell membrane constituents that bind PrV: a peripheral HSPG of high molecular weight, an integral HSPG of approximately 140 kDa, and an integral membrane protein of 85 kDa. It is tempting to speculate that interaction between PrV and the two species of HSPG mediates primary attachment of PrV and that the 85-kDa protein is involved in a subsequent attachment step.

Bovine herpesvirus 1 U(s) open reading frame 4 encodes a glycoproteoglycan.

Sequence analysis of the short unique (Us) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the Us open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the Us ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the Us ORF3 and Us ORF4, whereas the 1.8-kb RNA constitutes the monocistronic Us ORF4 mRNA. To identify the predicted BHV-I gG, recombinant vaccinia virus expressing the Us ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61- and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the Us ORF4 deletion mutant BHV-l/gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N- and O-linked carbohydrates, and that the high-molecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan C (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-I gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino-glycosylation, that has not yet been described for a herpesvirus-encoded protein.

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 x 10(-18) mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images.