Impact of COVID-19 vaccination on parental and childhood stress levels in Greece.

OBJECTIVE: The impact of COVID-19 vaccination on parental and childhood stress levels has not been thoroughly investigated. Our aim was to explore the above relationship and identify factors that may influence the dissemination of stress within the family during the pandemic. SUBJECTS AND METHODS: A cross-sectional e-survey was conducted among a nationwide sample of parents in May 2021 in Greece. Parental stress was assessed using the Perceived Stress Scale (PSS) and the Revised Impact of Event Scale (IES-R) tools. Childhood mental well-being was evaluated with the Children's Revised Impact of Event 13 (CRIES 13) scale. RESULTS: 1,703 unique questionnaires were analyzed; 19.5% of responders were completely vaccinated, 23.7% were partially vaccinated, 38.3% were awaiting vaccination, and 18.5% were classified as vaccine-hesitant (15.2% would delay, and 3.3% refused the vaccination). Stress levels were significantly lower in completely or partially vaccinated parents than in vaccine-hesitant ones (p<0.001 for PSS/IES-R). Vaccination status emerged as a strong and independent predictor of PSS and IES-R. A significant decrease in PSS and IES-R scores was observed in 991 participants between March 2020 and May 2021 (p<0.001 for PSS/IES-R). Vaccine uptake was associated with lower PSS and IES-R scores, irrespective of the phase of the pandemic or other sociodemographic factors. The CRIES 13 score of the participant's children (n=2,969) was 19.4 ±14.9 and positively correlated with the PSS and IES-R scores. Children whose parents were vaccinated had lower stress levels than those of vaccine-hesitant parents (p<0.001). CONCLUSIONS: Parental vaccination against COVID-19 is a significant stress and anxiety predictor for both parents and their offspring. Parental and childhood stress levels were correlated, while the effect of vaccination was independent of the pandemic phase. The campaigns to promote vaccine uptake against COVID-19 should also highlight its potential benefit on the psychological well-being of the family.

Child abuse experience, training, knowledge, and attitude of healthcare professionals in sixty hospitals in Greece.

OBJECTIVE: This study aims to record the overall perception of healthcare professionals on child abuse and identify potential affecting factors in a nationwide scale in Greece as well as to provide information that might be useful for future educational actions. MATERIALS AND METHODS: A total of 1,185 healthcare professionals in 60 hospitals with pediatric departments across Greece participated in this cross-sectional study. Participants included pediatricians, pediatric surgeons, residents, nurses, psychiatrists, psychologists, and social workers. Sections under investigation involved experience and training in child abuse, knowledge of formal and judicial issues, clinical knowledge, and self-assessment. RESULTS: Although more than half of the participants had confronted child abuse (n=712, 60.08%), only 273 (38.34% of them) submitted reports. One third of participants reported that they had received some training (n=440, 37.13%), mainly of postgraduate nature and based on personal initiative. Of those who reported child abuse, 175 (64.10%) had been trained. Each professional category was aware of topics regarding its own interest, without adequate knowledge of other disciplines. One third of psychiatrists, psychologists, and social workers felt confident in discussing with children and parents. Relevant scores were lower in the other categories. The lower scores were recorded among nurses and residents. The training deficit and reluctance to engage with judicial issues were the main causes of avoidance to deal with child abuse. CONCLUSIONS: Focused and organized training in child abuse is crucial to create reliable professionals in the field. The internet is a considerably helpful tool. Professionalism must characterize knowledge and practice in child abuse at the same level as in other medical topics. Motivation to engage should be early inspired and developed during the graduate years.

A novel presentation of an ATP1A3 gene mutation - case report and literature review.

OBJECTIVE: Mutations in the ATP1A3 gene cause the classical disorders of rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC) and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS). However, intermediate phenotypes have also been described, making the range of clinical manifestations associated with mutations in the ATP1A3 gene wider. A rare case of an ATP1A3 gene mutation is presented. CASE REPORT: Genetic testing was performed in a neonate who presented with neurological abnormalities on day 2 of life, severe electrolytic disturbances a few days later and developmental delay and epilepsy a few months later. A pathogenic heterozygous missense mutation in the ATP1A3 gene (c.2482G>A, E828K(p.Glu828Lys) was detected on clinical exome sequencing. CONCLUSIONS: The present case report extends the already described phenotypic variation observed in individuals with ATP1A3 gene mutations. It also illustrates the importance of genetic testing in the case of complex and not straightforward clinical scenarios, particularly when present from a very young age, before clinical criteria for known diagnoses are met.

Prader-Willi Syndrome and PCSK1 mutation: a novel presentation of combined syndromic and monogenic obesity.

OBJECTIVE: Prader-Willi syndrome (PWS) is a genomic imprinting disorder predominantly caused by the absence of paternally expressed imprinted genes at chromosome 15q11.2-q13. The PCSK1 gene is vital for the processing of hypothalamic POMC to ACTH and α-MSH, leading to food intake suppression and increased energy expenditure. The aim of this study was to investigate whether our PWS patient had a defect in genes involved in the hypothalamic melanocortin-4 receptor (MC4R) pathway. PATIENTS AND METHODS: A 27-year-old Greek man with PWS presented to the Adult Endocrine Clinic with morbid obesity and hyperphagia. He also had obstructive sleep apnea, growth hormone deficiency, gonadal failure and metabolic disturbances. At 6 years of age, chromosomal testing confirmed PWS with a deletion in the q11q13 region of the long arm of paternal chromosome 15. RESULTS: At the age of 27 years, further genetic testing was conducted, and next generation sequencing revealed a PCSK1_pN221D_HET mutation which was confirmed by Sanger sequencing. CONCLUSIONS: Our findings suggest that different genetic abnormalities may be present in an individual with PWS and that patients with PWS may need to be investigated for PCSK1 mutations, as the finding may potentially offer a novel treatment perspective for them.

Impaired adipocyte glucose transport regulators in morbid obesity - Possible mechanisms contributing to metabolic dysfunction.

OBJECTIVE: Obesity is characterized by hypertrophy and pathological expansion of adipocytes with impaired insulin signaling causing insulin resistance (IR) and metabolic dysfunction. We recently reported decreased expression of glucose transporter-4 (GLUT4) in cultured adipocytes from visceral and abdominal subcutaneous fat depots from patients with morbid obesity and hyperinsulinemia (MOW) and with Type 2 diabetes (MODM). Subsequently, we wanted to study the molecular mechanisms of the glucose transport regulators, p85PI3K, Rab5 and Gapex5 in morbid obesity. PATIENTS AND METHODS: Primary in vitro adipocyte cultures were developed from surgical biopsies from visceral (Visc) and abdominal (Sub) and gluteal subcutaneous (Glut) fat depots from 20 lean adults and 36 adults with morbid obesity divided into two groups: 20 with MOW and 16 MODM). mRNA and protein expression (P) of p85PI3K, Rab5 and Gapex5 were studied with RT-PCR and Western Immunoblotting (WI), respectively. RESULTS: In Sub, the P of (1) p85PI3K and Gapex5 were increased in MODM and (2) Rab5 was decreased in MOW and MODM compared to the lean. In Glut, the P of p85PI3K, Rab5 and Gapex5 showed no difference between the lean and MODM. CONCLUSIONS: In Sub of MODM (1) reduced RAB5 may possibly contribute to IR and glucose transport dysfunction, (2) increased Gapex5 may be a response to decreased Rab5 in an attempt to increase glucose transport and (3) increased p85PI3K may enhance IR mediating lipid accumulation in MODM. In Glut of MODM, though, the expression of p85PI3K, Rab5 and Gapex5 seems to be similar to that found in lean individuals.

Different patterns of p16 immunoreactivity in cervical biopsies: correlation to lesion grade and HPV detection, with a review of the literature.

p16 is one extensively studied marker in gynecological pathology. However, its routine application in the diagnosis of squamous intraepithelial lesions of the uterine cervix may present difficulties for the general pathologist. The aim of the present study was to examine a series of 100 cervical biopsies/LEEP specimens, with detailed HPV-typing, for patterns of p16 immunoreactivity and possible correlations with morphology and HPV types. Four patterns of immunopositivity were recognized, according to the distribution of positively stained cells, and these correlated to lesion grade. A review of the pertinent literature concerning p16 immunoreactivity in squamous intraepithelial lesions and nonneoplastic epithelia of the uterine cervix is included in an effort to summarize the existing data and the remaining questions at both the practical and theoretical level.

Delayed presentation of seropositivity in pre-existent coeliac disease in patients with Type 1 diabetes mellitus: a possible co-occurrence?

OBJECTIVE: The co-occurrence of coeliac disease (CD) and type 1 diabetes mellitus (T1DM) is well described and is mainly explained by sharing of common pathogenic mechanisms, such as common high-risk human lymphocyte antigen (HLA) genotypes (DR-DQ). PATIENTS AND METHODS: We describe a 12-year-old female patient with T1DM who presented with prolonged and severe glucose dysregulation. Extensive investigations, including coeliac screen, were negative. RESULTS: 3 years after glucose dysregulation manifested, coeliac screen testing was positive and coeliac disease was confirmed with bowel biopsy. Compliance to a gluten-free diet resulted in improvement of glucose control and seronegativity 9 months post-diagnosis. CONCLUSIONS: This is the first case report describing delayed seropositivity of CD and suggests that CD enteropathy may precede positive serology and could cause severe glucose dysregulation in patients with T1DM.

The prolonged effects of COVID-19. A new "threat"?

Coronavirus 'long-haulers" currently represent a significant public health concern. Recent reports suggest that persistent effects of COVID-19, such as fatigue, dyspnea, chest pain, anxiety, depression, arthralgia, may last for months and lead to a decline in quality of life. Risk factors for long COVID are still not very well understood. Survivors suffer from ongoing symptoms. This new entity highlights the need for a multidisciplinary approach that would enable closer monitoring of affected patients and implementation of measures that could reduce the impact of the pandemic on the overall patient wellbeing after the resolution of acute symptoms.

Impact of COVID-19 on new-onset type 1 diabetes mellitus - A one-year prospective study.

OBJECTIVE: A positive relationship between the recently emerged Corona Virus Disease-19 (COVID-19) and diabetes has been inferred, but not confirmed, in children. The aim of the present study was to investigate the possible impact of COVID-19 on new-onset Type-1 Diabetes Mellitus (T1DM) in a pediatric population. PATIENTS AND METHODS: This is a prospective study of all children and adolescents diagnosed with T1DM during the first year of the COVID-19 pandemic (March 2020-February 2021) in Western Greece (population coverage ≈1,000,000). The incidence and severity of T1DM, the age and sex of the participants and HbA1c and c-peptide concentrations at diagnosis were recorded and compared to those of the previous year (pre-COVID-19 year). RESULTS: 21 children aged 8.03±0.90 years old were diagnosed with T1DM in the COVID-19 year and 17, aged 9.44±3.72 years old, in the pre-COVID-19 year. A different seasonality pattern of new onsets was observed during the COVID-19 year compared to the previous year, with increasing trend from spring to winter (spring: 9.5% vs. 23.5%, autumn: 23.8% vs. 29.4%, summer: 19% vs. 11.8%, winter: 47.6% vs. 35.3%). Also, compared to the preceding year, HbA1c was significantly higher (p=0.012) and the incidence and severity of diabetic ketoacidosis greater (p=0.045, p=0.013, respectively). CONCLUSIONS: This is the first study to report a different seasonality pattern and increased severity of new-onset T1DM during the first year of the COVID-19 pandemic. Future research should further investigate the possible role of SARS-CoV-2 and the different pattern of overall infection incidence during the COVID-19 year.

Alterations in lymphocyte subsets and monocytes in patients diagnosed with SARS-CoV-2 pneumonia: a mini review of the literature.

OBJECTIVE: Complete blood count parameters are frequently altered in COVID-19 patients. Leucopenia and lymphopenia are the most common findings. This is not specific to COVID-19 as similar alterations are found in various other viral infections. This work is intended to summarize the evidence regarding white blood cell and lymphocyte subset alterations in COVID-19 and their clinical implications. MATERIALS AND METHODS: A PubMed search was conducted to identify relevant original studies. Articles not available in English or referring exclusively to pediatric patients were excluded. The study was designed as a narrative review from its inception. RESULTS: Complete white blood cell number and lymphocytes may be reduced in COVID-19 patients. Circulating CD4+ cells (helper T lymphocytes), CD8+ cells (cytotoxic T lymphocytes), regulatory T cells and natural killer (NK) cells may be reduced, with a greater reduction observed in critically ill patients. CD4+ and regulatory cell deficiencies may contribute to the cytokine storm and subsequent tissue damage observed in severe COVID-19 infection. NK and CD8+ cell deficiency might delay infection clearance. These aberrations of cellular immunity may contribute significantly to the pathogenesis of the disease. Alterations observed in monocyte function can also be implicated as they are effector cells responsible for tissue damage and remodeling. B cell dysfunction and maturation abnormalities have also been reported, suggesting that the virus also impairs humoral immunity. CONCLUSIONS: Lymphocyte subset abnormalities may be useful prognostic biomarkers for COVID-19, with circulating CD8+ cell count being the most promising as a predictor of severe disease requiring mechanical ventilation and mortality.

Glucose tolerance and insulin sensitivity markers in children and adolescents with excess weight.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) and obesity are alarmingly increasing in children and adolescents. Hence, predictors for early metabolic abnormalities in childhood are urgently needed. We investigated glucose tolerance in children and adolescents with obesity, markers of insulin sensitivity between males and females and the potential association between the parameters measured during an OGTT (glucose, insulin, c-peptide) and prediabetes or stages of puberty. PATIENTS AND METHODS: Glucose tolerance in 89 children and adolescents with excess weight, aged 4-19 years, from Western Greece was studied. A 3-hour OGTT was performed and fasting glucose (FG), fasting insulin (FI), 1/FI, FG/FI, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), Quantitative insulin sensitivity check index (QUICKI), ISI Matsuda index and Insulinogenic index (IGI30), were also calculated. RESULTS: No significant differences were observed in glucose values between males and females. Insulin and c-peptide concentrations were higher in the girls at several time points. FG/FI was significantly higher in the boys. Girls with obesity may be at higher risk for future insulin resistance. CONCLUSIONS: Better surveillance of pubertal girls with obesity is crucial and can be achieved using additional information provided by an OGTT, since they appear to be at a higher risk for beta-cell exhaustion. During the OGTT, not only are the baseline and 2-hour glucose and insulin measurements useful for predicting future metabolic risks and development of T2DM in children and adolescents with obesity, but additional time measurements may also be helpful.

The association between obesity and the risk for development of eating disorders - A large-scale epidemiological study.

OBJECTIVE: Eating disorders and obesity are serious, multifactorial diseases with increasing prevalence worldwide, often manifesting during childhood and adolescence. The aim of this study was to investigate the risk for developing eating disorders in children and adolescents of Primary and Secondary Education, with normal or excessive body weight. MATERIALS AND METHODS: A representative sample (N=3504) of students from schools of Western Greece, 50.2% boys, aged 10-16 years old, participated in the present cross-sectional epidemiological study. The students' dietary habits were assessed through multiple-choice questions and the risk for the development of eating disorders was evaluated using the Eating Attitudes Scale (EAT-13) validated questionnaire. Anthropometric measurements were obtained and the BMI, BMI% and BMI z-score were calculated. RESULTS: Nearly 20% of the participants, particularly those with overweight or obesity, were at increased risk for developing eating disorders (25% of normal weight-, 28.2% of overweight- and 33% of participants with obesity). Boys were more likely to develop eating disorders than girls, but not statistically significantly. A positive correlation of: (1) the overall EAT-13 score, (2) food pre-occupation score and (3) dieting score, with BMI z-score and obesity was found, as opposed to a negative correlation of Important Others score with BMI z-score and obesity. CONCLUSIONS: Increased awareness regarding the risk for developing eating disorders or disordered eating in children and adolescents with overweight and obesity is recommended to avoid underdiagnosis of this condition. Prompt identification of children at risk contributes to the implementation of targeted and effective prevention and treatment interventions.

Immature teratoma in pregnancy: a case report and literature review.

BACKGROUND: The management of a Stage I immature teratoma during pregnancy with a review of the literature is reported. CASE REPORT: A growing adnexal mass was removed at 12 weeks of gestation. Although the frozen section was negative, because of intraoperative clinical suspicion, a right salpingo-oophorectomy and surgical staging were performed. Histological examination revealed a Stage Ia, grade 1 immature ovarian teratoma. Appropriate surgical staging enabled avoidance of chemotherapy despite the unexpected histological diagnosis. The pregnancy was terminated because of fetal distress, with cesarean section at 34 weeks of gestation. At that time the peritoneal cavity was inspected and biopsies were taken as in second-look laparotomy. Two years after the first operation the patient remains disease free. CONCLUSION: For adnexal masses removed during pregnancy frozen section is useful but when there is clinical suspicion surgical staging must be performed.

Fascin can be an auxiliary immunomarker of ovarian granulosa cell tumors: comparison with calretinin and inhibin-alpha.

The histopathologic diagnosis of granulosa cell tumor adult type (AGCT) can be supported by the use of established immunomarkers such as inhibin-alpha and calretinin. Previously unreported data is presented on the detection of fascin in AGCT, in nonneoplastic granulosa cells and in other types of sex-cord stromal tumors. In addition, by staining a panel of various tumors, potentially included in the differential diagnosis of AGCT, we assessed the value of fascin as an auxiliary AGCT immunomarker. Intense and strong fascin staining may assist in cases with ambiguous calretinin or inhibin-alpha staining. On the contrary, absence of fascin should question a provisional morphologic diagnosis of AGCT.

L1 (CAM) (CD171) in ovarian serous neoplasms.

PURPOSE OF THE INVESTIGATION: The evaluation of L1 (CAM) as a tumor progression marker and as a prognostic factor in serous ovarian tumors. METHODS: L1 (CAM) protein expression was assessed by immunohistochemistry and Western blot in serous ovarian tumors [cystadenomas (n = 20), borderline tumors (n = 14) and carcinomas (n = 47)], and was correlated with stage,grade, progression-free survival time (PFS) and overall survival. RESULTS: L1 (CAM) immunoreactivity correlated significantly with stage and grade. It increased from benign tumors to early carcinomas and to advanced stage carcinomas progressively and significantly. In Stage III G3 carcinoma patients, low L1 (CAM) expressing tumors exhibited better response to chemotherapy and were associated with statistically significantly longer PFS (p = 0.002). CONCLUSION: L1 (CAM) expression represents a novel diagnostic marker in serous ovarian neoplasms that shows characteristics of tumor progression. L1 expression was associated with chemotherapy response.

Xanthogranulomatous salpingitis: report of three cases and comparison with a case of pseudoxanthomatous salpingitis.

Xanthogranulomatous inflammation, an uncommon form of chronic inflammation, has been described in several organs including those of the female genital tract. A rare condition described as pseudoxanthomatous salpingitis or pseudoxanthomatous salpingiosis, which is often associated with endometriosis, has been distinguished from xanthogranulomatous inflammation of the fallopian tube based on its histological features. In the present report three cases of xanthogranulomatous salpingitis and one case of pseudoxanthomatous salpingitis are presented and their clinical, pathological and histochemical features are compared.

Retinoid receptor alpha and Beta expression in serous ovarian tumors.

The expression of retinoid acid receptors alpha (RARalpha) and beta (RARbeta) and estrogen receptor alpha (ERalpha) was assessed by immunohistochemistry and Western blotting in normal ovaries, serous cystadenoma (n = 20), serous borderline (n = 14), and serous ovarian cancer (n = 47) and was correlated in cancer cases with stage, grade, progress-free survival (PFS), and survival. RARalpha was increasingly expressed in benign cystadenomas, borderline, and low-stage and advanced-stage neoplasms (p < 0.001). In stage III, G3 serous carcinoma, increased RARalpha expression was an independent prognostic factor associated with lower chemoresponse to first-line chemotherapy (taxol and carboplatin) and shorter PFS (p < 0.002).RARbeta and ERalpha expression did not correlate with RARalpha tumor characteristics or PFS and survival.

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.

The role of human telomerase catalytic subunit mRNA expression in cervical dysplasias.

Telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression were investigated in cervical specimens and were correlated with cytologic findings and the presence of human papilloma virus (HPV) infection. Telomerase activity was evaluated by the telomeric repeat protocol assay and hTERT mRNA expression was evaluated by reverse transcriptase polymerase chain reaction (PCR). HPV DNA was detected by PCR, as well as restriction endonuclease digestion. HPV DNA was detected in all 82 specimens with abnormal cytologic findings and in 4 of 34 normal samples. Low-grade squamous intraepithelial lesions (LGSILs) were present in 74 of 82 specimens (90.2%) and high-grade squamous intraepithelial lesions (HGSILs) were present in 8 of 82 (9.75%) specimens. Seven of the eight HGSIL (87.5%) and 26 of 74 LGSIL (35.1%) specimens were hTERT positive, whereas all normal specimens were hTERT mRNA negative. Telomerase activity was detected in 21 of 74 (28.4%) LGSIL/atypical squamous epithelial cells of undetermined significance (ASCUS) and in five of eight (62.5%) HGSIL samples. A correlation was observed among telomerase activity, hTERT mRNA expression, and high-risk HPV infection in HGSIL samples (P < 0.001). High-risk HPV infection assessment showed 75% sensitivity and 72.2% specificity for HGSILs. Telomerase activity assessment in cervical smears showed sensitivity and negative predictive value (NPV) for HGSILs 62.5% and 96.7%, whereas specificity and positive predictive value (PPV) were 80.5% and 19.2%, respectively. hTERT mRNA expression assessment showed 87.5% sensitivity and 98.7% NPV for HGSILs, whereas specificity and PPV were 76% and 21.2%, respectively. Based on the above-described telomerase assessment values, it is suggested that the telomerase system might not be an appropriate diagnostic marker for cytology, given that the final evaluation must rely on a combination of all available test assessment data, clinical diagnosis, as well as the follow-up of all LGSIL samples that were positive for telomerase activation.

2D-gel spot detection and segmentation based on modified image-aware grow-cut and regional intensity information.

BACKGROUND: Proteomics, the study of proteomes, has been increasingly utilized in a wide variety of biological problems. The Two-Dimensional Gel Electrophoresis (2D-PAGE) technique is a powerful proteomics technique aiming at separation of the complex protein mixtures. Spot detection and segmentation are fundamental components of 2D-gel image analysis but remain arduous and difficult tasks. Several software packages and academic approaches are available for 2D-gel image spot detection and segmentation. Each one has its respective advantages and disadvantages and achieves a different level of success in dealing with the challenges of 2D-gel image analysis. A common characteristic of the available methods is their dependency on user intervention in order to achieve optimal results, a process that can lead to subjective and non-reproducible results. In this work, the authors propose a novel spot detection and segmentation methodology for 2D-gel images. METHODS: This work introduces a novel spot detection and spot segmentation methodology that is based on a multi-thresholding scheme applied on overlapping regions of the image, a custom grow-cut algorithm, a region growing scheme and morphological operators. The performance of the proposed methodology is evaluated on real as well as synthetic 2D-gel images using well established statistical measures, including precision, sensitivity, and their weighted measure, F-measure, as well as volumetric overlap, volumetric error and volumetric overlap error. RESULTS: Experimental results show that the proposed methodology outperforms state-of-the-art software packages and methods proposed in the literature and results in more plausible spot boundaries and more accurate segmentation. The proposed method achieved the highest F-measure (94.8%) for spot detection and the lowest volumetric overlap error (8.3%) for the segmentation process. CONCLUSIONS: Evaluation against state-of-the-art 2D-gel image analysis software packages and techniques proposed in the literature, including Melanie 7, Delta2D, PDQuest and Scimo, demonstrates that the proposed approach outperforms the other methods evaluated in this work and constitutes an advantageous and reliable solution for 2D-gel image analysis.