Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

Biomarkers of immune capacity, infection and inflammation are associated with poor outcome and mortality after stroke - the PREDICT study.

BACKGROUND: Almost 40% of stroke patients have a poor outcome at 3 months after the index event. Predictors for stroke outcome in the early acute phase may help to tailor stroke treatment. Infection and inflammation are considered to influence stroke outcome. METHODS: In a prospective multicenter study in Germany and Spain, including 486 patients with acute ischemic stroke, we used multivariable regression analysis to investigate the association of poor outcome with monocytic HLA-DR (mHLA-DR) expression, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide-binding protein (LBP) as markers for immunodepression, inflammation and infection. Outcome was assessed at 3 months after stroke via a structured telephone interview using the modified Rankin Scale (mRS). Poor outcome was defined as a mRS score of 3 or higher which included death. Furthermore, a time-to-event analysis for death within 3 months was performed. RESULTS: Three-month outcome data was available for 391 patients. Female sex, older age, diabetes mellitus, atrial fibrillation, stroke-associated pneumonia (SAP) and higher National Institute of Health Stroke Scale (NIHSS) score as well as lower mHLA-DR levels, higher IL-6 and LBP-levels at day 1 were associated with poor outcome at 3 months in bivariate analysis. Furthermore, multivariable analysis revealed that lower mHLA-DR expression was associated with poor outcome. Female sex, older age, atrial fibrillation, SAP, higher NIHSS score, lower mHLA-DR expression and higher IL-6 levels were associated with shorter survival time in bivariate analysis. In multivariable analysis, SAP and higher IL-6 levels on day 1 were associated with shorter survival time. CONCLUSIONS: SAP, lower mHLA-DR-expression and higher IL-6 levels on day one are associated with poor outcome and shorter survival time at 3 months after stroke onset. TRIAL REGISTRATION: www.clinicaltrials.gov, NCT01079728 , March 3, 2010.

ENerGetIcs in hypertrophic cardiomyopathy: traNslation between MRI, PET and cardiac myofilament function (ENGINE study).

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease mostly due to mutations in genes encoding sarcomeric proteins. HCM is characterised by asymmetric hypertrophy of the left ventricle (LV) in the absence of another cardiac or systemic disease. At present it lacks specific treatment to prevent or reverse cardiac dysfunction and hypertrophy in mutation carriers and HCM patients. Previous studies have indicated that sarcomere mutations increase energetic costs of cardiac contraction and cause myocardial dysfunction and hypertrophy. By using a translational approach, we aim to determine to what extent disturbances of myocardial energy metabolism underlie disease progression in HCM. METHODS: Hypertrophic obstructive cardiomyopathy (HOCM) patients and aortic valve stenosis (AVS) patients will undergo a positron emission tomography (PET) with acetate and cardiovascular magnetic resonance imaging (CMR) with tissue tagging before and 4 months after myectomy surgery or aortic valve replacement + septal biopsy. Myectomy tissue or septal biopsy will be used to determine efficiency of sarcomere contraction in-vitro, and results will be compared with in-vivo cardiac performance. Healthy subjects and non-hypertrophic HCM mutation carriers will serve as a control group. ENDPOINTS: Our study will reveal whether perturbations in cardiac energetics deteriorate during disease progression in HCM and whether these changes are attributed to cardiac remodelling or the presence of a sarcomere mutation per se. In-vitro studies in hypertrophied cardiac muscle from HOCM and AVS patients will establish whether sarcomere mutations increase ATP consumption of sarcomeres in human myocardium. Our follow-up imaging study in HOCM and AVS patients will reveal whether impaired cardiac energetics are restored by cardiac surgery.

Chip calorimetry for fast and reliable evaluation of bactericidal and bacteriostatic treatments of biofilms.

Chip calorimetry is introduced as a new monitoring tool that provides real-time information about the physiological state of biofilms. Its potential for use for the study of the effects of antibiotics and other biocides was tested. Established Pseudomonas putida biofilms were exposed to substances known to cause toxicity by different mechanisms and to provoke different responses of defense and resistance. The effects of these compounds on heat production rates were monitored and compared with the effects of these compounds on the numbers of CFU and intracellular ATP contents. The real-time monitoring potential of chip calorimetry was successfully demonstrated by using as examples the fast-acting poisons formaldehyde and 2,4-dinitrophenol (DNP). A dosage of antibiotics initially increased the heat production rate. This was discussed as being the effect of energy-dependent resistance mechanisms (e.g., export and/or transformation of the antibiotic). The subsequent reduction in the heat production rate was attributed to the loss of activity and the death of the biofilm bacteria. The shapes of the death curves were in agreement with the assumed variation in the levels of exposure of cells within the multilayer biofilms. The new monitoring tool provides fast, quantitative, and mechanistic insights into the acute and chronic effects of a compound on biofilm activity while requiring only minute quantities of the biocide.

Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Diversity of the basic defect of homozygous CFTR mutation genotypes in humans.

BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

Terminal deoxynucleotidyl transferase-positive cells in trephine biopsies following bone marrow or peripheral stem cell transplantation reflect vigorous B-cell generation.

AIMS: Bone marrow is the major site of B-cell generation in humans. While in early childhood a high number of B-cell precursors is found in the bone marrow, only very few such cells are usually detectable in adult bone marrow. To assess the number of immature B cells present after haematopoietic cell transplantation the number of terminal deoxynucleotidyl transferase (TdT)-positive cells in regenerating bone marrow of adult patients was analysed. METHODS AND RESULTS: Bone marrow biopsy specimens were analysed from patients after allogeneic bone marrow transplantation (BMT; n = 14) or stem cell transplantation (SCT; n = 25) and autologous BMT (n = 9). Specimens from 11 untransplanted adult patients and 11 infants were also studied, as negative and positive controls, respectively. Immunohistochemistry was performed on paraffin-embedded bone marrow biopsy sections using TdT as a marker of lymphoid progenitors. Immunoreactivity for CD79a, CD20 and CD10 was used to confirm their B-cell origin. Using computer-assisted automated image analysis we quantitatively assessed the TdT+ cells present. We found a significant increase in the numbers of B-cell precursors in the bone marrow after allogeneic and autologous BMT/SCT compared with adult controls (P = 0.022). To analyse this in detail, we followed some patients after allogeneic BMT/SCT for up to 1445 days, when a marked B-cell increase was still detectable. However, the median number of TdT+ B cells after BMT/SCT was significantly lower than the number of equivalent B cells in infantile bone marrow biopsy specimens (P < 0.001). CONCLUSIONS: Bone marrow of adult patients after BMT/SCT is capable of initiating vigorous precursor B-cell generation, which is not seen in untransplanted adults. However, the increase of immature B cells was variable in our study. Only in two young adult patients did it reach the magnitude of B-cell generation seen in infantile bone marrow where immunocompetent B cells are produced normally. A marked increase in number of immature B cells post-transplant may mimic B-cell acute lymphoblastic leukaemia (B-ALL). This is a potential problem in patients transplanted for B-ALL itself. Since reactive and neoplastic B-cell precursors share the same immunophenotype in paraffin-embedded tissue, additional tools, particularly molecular techniques, may have to be employed to establish the correct diagnosis.

Dornase alfa in the treatment of cystic fibrosis in Europe: a report from the Epidemiologic Registry of Cystic Fibrosis.

Dornase alfa (Pulmozyme) treatment for patients with cystic fibrosis (CF) has been shown to improve pulmonary function and reduce exacerbations of infection in a number of placebo-controlled double-blind studies. Data in the Epidemiologic Registry of Cystic Fibrosis (ERCF) in November 1998 were used to assess the long-term effectiveness in routine clinical practice of dornase alfa in terms of pulmonary function and frequency of acute pulmonary exacerbations in CF. At that time, the ERCF contained data on 13,684 CF patients, with a mean observation period of 2.3 years. To be included in the analysis, patients had to have 2 years of data in the Registry in appropriate detail. Overall, untreated patients showed a decline in forced expiratory volume in 1 sec over a 2-year period of -2.3% predicted, but treated patients were stable, showing a change of 0.3% predicted, i.e., a treatment benefit of 2.5%. Compared to untreated patients, there were 25 fewer exacerbations per 100 treated patients per year. The analysis suggested that younger patients were likely to benefit more from treatment. The findings of randomized clinical trials were supported by the data collected in routine clinical practice.

Presence of cystic fibrosis-related diabetes mellitus is tightly linked to poor lung function in patients with cystic fibrosis: data from the European Epidemiologic Registry of Cystic Fibrosis.

Data derived from a cross-sectional analysis of 7,566 patients stratified into six age groups were used to compare lung function, body mass index (BMI), and weight for age in patients with and without cystic fibrosis-related diabetes mellitus (CFDM). The presence of CFDM was tightly linked to poor lung function, regardless of age. The mean value of FEV(1) % predicted in the age groups < 10, 10-< 15, 15-< 20, 20-< 25, 25-< 30, and 30 years or older were 87%, 77%, 69%, 58%, 55%, and 53% in the nondiabetic cystic fibrosis (CF) patients as compared to 79%, 66%, 55%, 49%, 46%, and 44% in the diabetic CF patients. BMI and weight for age were also lower in diabetic than nondiabetic CF patients in all age groups, except for BMI in the youngest patients. The difference in lung function and in nutritional parameters between diabetic and nondiabetic CF patients was not linked to presence or absence of any specific pathogen in the lower respiratory tract. These results confirm and extend those of earlier studies in smaller numbers of patients, and they clearly identify CFDM as a powerful determinant of severe lung disease and reduced survival in patients with CF and diabetes mellitus.

Factors associated with poor pulmonary function: cross-sectional analysis of data from the ERCF. European Epidemiologic Registry of Cystic Fibrosis.

The European Epidemiologic Registry of Cystic Fibrosis began collecting longitudinal data on European cystic fibrosis patients in 1994. A cross-sectional analysis was performed to identify the factors associated with low values of % predicted forced expiratory volume in one second (FEV1) upon patient enrollment. Data from 7,010 patients aged > or =6 yrs were included. Clinical conditions, microbiological isolates and medications reported at enrollment or within the following 180 days were analysed for age-specific associations. Factors associated with FEV1 that were lower by >10% of pred values were: lower weight for age percentiles, haemoptysis, pneumothorax, pulmonary symptoms at presentation, Pseudomonas aeruginosa, Burkholderia cepacia, oral corticosteroids, nonsteroid anti-inflammatory drugs, dornase alfa, oxygen and assisted ventilation and, in patients >12 yrs old only, use of airway clearance techniques, inhaled bronchodilators, oral nutritional supplements, pancreatic enzymes and insulin or oral hypoglycaemics. Slightly impaired lung function (5-10%) was associated with: diabetes (> or = 18-yrs-old), gastro-oesophageal reflux, allergic bronchopulmonary aspergillosis, asthma-like symptoms, portal hypertension, Aspergillus spp. and Candida spp. Sex, Haemophilus influenzae and Staphylococcus aureus were not associated with impaired pulmonary status. Regular exercise (especially in older patients) and nasal polyposis were associated with slightly better FEV1. The results confirm those of previous studies and suggest selective prescribing in sicker patients.

European Epidemiologic Registry of Cystic Fibrosis (ERCF): comparison of major disease manifestations between patients with different classes of mutations.

SUMMARY. By August 1997, 11,749 patients with cystic fibrosis had been enrolled in the European Epidemiologic Registry of Cystic Fibrosis (ERCF). Genotype analysis had been performed on 8,963 (76%) of these patients, and the majority had one or two identifiable mutations. Patients with known mutations were classified according to the type of mutation (Classes I-V), and were grouped according to the class of mutation on both chromosomes. This resulted in six subgroups, including all patients homozygous for Class I (I/I, n = 72), for Class II (II/II, n = 5,020), and for Class III mutations, (III/III, n = 23). Since there were only 23 patients homozygous for Class III mutations, a fourth group was made up of patients who were compound heterozygous for a Class II and III mutation (II/III, n = 265). There were only five patients homozygous for Class IV mutations, and consequently a fifth group was made up of all patients carrying at least one Class IV mutation, regardless of the nature of the mutation on the other chromosome (IV/any, n = 187). None were homozygous for Class V mutations; consequently, a sixth group consisted of patients carrying at least one Class V mutation (V/any, n = 22). Mean age was highest in groups III/III, IV/any, and V/any (15.6, 16, and 17 years, respectively) as opposed to 12.4 years in group II/II and 13.4 in group II/III, but both group III/III and V/any were small, and the confidence interval of the mean was large. The percentage of patients receiving pancreatic enzymes was lower in groups IV/any and V/any than in any of the other groups, i.e., approximately 50% of patients 18 years or older in both groups as opposed to between 90-100% of all other patients regardless of age. The prevalence of diabetes mellitus increased with age from 2.6% in patients < 18 years to 22.1% in patients 18 years or older in the large group II/II, but was only 1.5% in patients 18 years or older in group IV/any. Disregarding the small group III/III, abnormally elevated liver enzymes and/or bilirubin (1.5 x upper normal limit) was much less frequent in group IV/any than in any of the other groups, both overall and in patients aged 18 years or more. The course of lung disease appeared to be less dependent on genotype than pancreatic function, with only minor differences between groups; however, the mean values of both FVC % and FEV(1) % were slightly higher in group IV/any than all other groups in both younger and older patients. The same was found for the prevalence of some major clinical signs of severe lung disease, such as clubbing, hyperinflation, and crepitations. Overall mean weight expressed as an age percentile was markedly higher in group IV/any than in any other group, which may be related to the finding of a much lower prevalence of chronic P. aeruginosa infection in patients 18 years or older belonging to group IV/any (and V/any) than in any other group. In conclusion, the presence of a class IV mutation appears to offer some degree of protection against pancreatic insufficiency, diabetes mellitus, and liver disease. We confirmed that lung disease follows a milder clinical course in patients with a class IV mutation and that the presence of a class IV mutation (and possibly class V) is associated with a delay in the onset of P. aeruginosa infection.

Allergic bronchopulmonary aspergillosis in cystic fibrosis. A European epidemiological study. Epidemiologic Registry of Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a disease resulting from a hypersensitivity response to Aspergillus fumigatus, although the pathogenesis of the disease is unknown and its prevalence in cystic fibrosis (CF) is still poorly defined. Data from the Epidemiologic Registry of Cystic Fibrosis (ERCF) on 12,447 CF patients gathered from 224 CF centres in nine European countries were analysed. The ERCF definition of ABPA diagnosis is a positive skin test and serum precipitins to A. fumigatus, together with serum immunoglobulin (Ig)E levels >1,000 U x mL(-1) and additional clinical or laboratory parameters. The overall prevalence of ABPA in the ERCF population was 7.8% (range: 2.1% in Sweden to 13.6% in Belgium). Prevalence was low <6 yrs of age but was almost constant approximately 10% thereafter. No sex differences were observed. ABPA affected 8.0% of patients with a deltaF508/deltaF508 genotype and 5-6% with deltaF508/G551D, deltaF508/G542X and deltaF508/N1303K genotypes. ABPA patients presented a lower forced expiratory volume in one second (FEV1) than those without ABPA at any age and the prevalence ranged from 6.6% in patients with FEV1 > or =20-12.9% in those with FEV1 <40%. ABPA was associated with higher rates of microbial colonization, pneumothorax and massive haemoptysis, and with higher IgG serum levels and poorer nutritional status. A mixed model regression analysis of lung function showed that FEVI decline during the follow-up period was not substantially different in ABPA patients compared with non-ABPA patients for any subgroups based on age or disease severity at enrollment. To conclude, allergic bronchopulmonary aspergillosis is a frequent complication in cystic fibrosis patients, particularly after the age of 6 yrs, and it is generally associated with a poorer clinical condition. However, any clear independent influence of allergic bronchopulmonary aspergillosis on the rate of lung function decline in the short term was not shown.

Bacterial phosphating of mild (unalloyed) steel.

Mild (unalloyed) steel electrodes were incubated in phosphate-buffered cultures of aerobic, biofilm-forming Rhodococcus sp. strain C125 and Pseudomonas putida mt2. A resulting surface reaction leading to the formation of a corrosion-inhibiting vivianite layer was accompanied by a characteristic electrochemical potential (E) curve. First, E increased slightly due to the interaction of phosphate with the iron oxides covering the steel surface. Subsequently, E decreased rapidly and after 1 day reached -510 mV, the potential of free iron, indicating the removal of the iron oxides. At this point, only scattered patches of bacteria covered the surface. A surface reaction, in which iron was released and vivianite precipitated, started. E remained at -510 mV for about 2 days, during which the vivianite layer grew steadily. Thereafter, E increased markedly to the initial value, and the release of iron stopped. Changes in E and formation of vivianite were results of bacterial activity, with oxygen consumption by the biofilm being the driving force. These findings indicate that biofilms may protect steel surfaces and might be used as an alternative method to combat corrosion.

Myoepithelial sialadenitis versus low-grade non-Hodgkin's lymphoma of the salivary gland in FNAB: is discrimination by means of an image processing system possible?

The diagnosis of myoepithelial sialadenitis (MESA) in fine needle aspiration biopsy may be difficult. There is a dense lymphocytic infiltration in the gland and discrimination between a hyperimmune reaction and a low grade non-Hodgkin's lymphoma (NHL) of B-cell origin may be impossible. To get additional diagnostically helpful criteria, texture feature analysis on routinely obtained FNAB's of the salivary gland was applied. In the data set 36/36 cases of low grade B-NHL confirmed by histology and 10/13 histologically confirmed cases of MESA could be classified correctly by means of an image processing system. The chromatin structure of each nucleus was classified by texture features (n = 6), which were determined according to the method of Harms et al. For statistical analysis of the cell types a classification tree based on the commercial program CART was applied. The data set of 49 cases was proved by the crossvalidation test 10 fold. The calculated diagnosis for each case suggests that this method may be helpful in the cytologically doubtful cases.

Frequency of trisomy 20 in nonmalignant bronchial epithelium from lung cancer patients and cancer-free former uranium miners and smokers.

Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.

Multicenter, open-label study of recombinant human DNase in cystic fibrosis patients with moderate lung disease. DNase International Study Group.

Cystic fibrosis is characterized by the accumulation of thick viscous purulent secretions. Recombinant human deoxyribonuclease I (rhDNase) breaks down extracellular DNA, which contributes to the increased viscosity of sputum. A multinational, open-label study was conducted in 974 cystic fibrosis patients with moderate lung disease [forced vital capacity (FVC) 40-70% of predicted values] to examine the safety and efficacy of aerosolized rhDNase, 2.5 mg, once daily over a period of at least 12 weeks. Patients were assessed under conditions reflecting routine clinical practice. During rhDNase therapy, at least one respiratory tract infection (RTI) requiring intravenous antibiotics was experienced by 29.5% of patients. Forced expiratory volume in 1 second (FEV1) and FVC were significantly improved from baseline by a mean of 10.5% and 7.2%, respectively. Voice alteration and pharyngitis were the most frequent rhDNase-related adverse events, but only 2% of all patients discontinued treatment due to adverse events. The results obtained were similar to a subanalysis of data from the first 3 months of a placebo-controlled U.S. study. The patients in the present study had a similar frequency of RTIs and improvement in pulmonary function, and reported fewer rhDNase-related and cystic fibrosis-related adverse events than patients in the U.S. study. We conclude that administration of rhDNase is safe, well tolerated, and effective under conditions reflecting routine clinical practice in patients with cystic fibrosis and moderate lung disease.

Differentiation of low-grade non-Hodgkin's lymphomas using paraffin sections by image processing.

In a previous study, we were able to demonstrate that the differentiation of low-grade non-Hodgkin's lymphomas (NHLs) using digital image analysis of touch imprints obtained from native tumor tissue is feasible. The availability of touch imprints in routine diagnostics, however, is restricted. Therefore, we extended our studies toward paraffin sections being used as routine material for histological diagnoses. To identify five types of NHL classified according to the Revised European American Lymphoma classification, paraffin sections (n=53) of NHL and 9 reactive lymphoid tissues (RLTs) were scanned with a color-video-based microscope system and analyzed by digital image processing. A reliable division between benign and neoplastic lymphoproliferations was achieved. We were able to identify 78% of RLTs as benign and 94% of NHLs as neoplastic. The average probability of correct identification into the six subgroups was 66%. In detail, 78% of RLTs, 50% of chronic lymphocytic lymphomas and MALT-type lymphomas, 72% of mantle cell lymphomas, and 67% of follicle center cell lymphoma were classified correctly. Although the method of subclassifying or identifying NHLs on the basis of a computer-mediated assay is still not usable in daily practice, we show that a reliable differentiation between reactive and neoplastic lymphoproliferative lesions can be achieved by analysis of paraffin sections with high-resolution image analysis and that it is possible to define nuclear structures by identifying subtypes of NHL.