What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes.

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Molecular Diagnostic Tools against SARS-CoV-2 in Poland in 2022.

The most effective way to stop the spread of COVID-19 (coronavirus disease 2019) is to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and isolate those infected as soon as possible. More than 1000 types of molecular and antigen-based immunoassay tests to detect SARS-CoV-2 are now commercially available worldwide. In this review, we present the possibilities of molecular diagnostics available in Poland in 2022. We provide a description of what samples have proven useful to confirm SARS-CoV-2 infection, we describe what methods are used, as well as what safeguards can and should be used to prevent false-negative and false-positive results, and finally we review the products that diagnostic laboratories have to choose from. We also describe diagnostic problems associated with the mutation of the virus.

Predictive performance and clinical application of COV50, a urinary proteomic biomarker in early COVID-19 infection: a prospective multicentre cohort study.

BACKGROUND: The SARS-CoV-2 pandemic is a worldwide challenge. The CRIT-CoV-U pilot study generated a urinary proteomic biomarker consisting of 50 peptides (COV50), which predicted death and disease progression from SARS-CoV-2. After the interim analysis presented for the German Government, here, we aimed to analyse the full dataset to consolidate the findings and propose potential clinical applications of this biomarker. METHODS: CRIT-CoV-U was a prospective multicentre cohort study. In eight European countries (Austria, France, Germany, Greece, North Macedonia, Poland, Spain, and Sweden), 1012 adults with PCR-confirmed COVID-19 were followed up for death and progression along the 8-point WHO scale. Capillary electrophoresis coupled with mass spectrometry was used for urinary proteomic profiling. Statistical methods included logistic regression and receiver operating characteristic curve analysis with a comparison of the area under curve (AUC) between nested models. Hospitalisation costs were derived from the care facility corresponding with the Markov chain probability of reaching WHO scores ranging from 3 to 8 and flat-rate hospitalisation costs adjusted for the gross per capita domestic product of each country. FINDINGS: From June 30 to Nov 19, 2020, 228 participants were recruited, and from April 30, 2020, to April 14, 2021, 784 participants were recruited, resulting in a total of 1012 participants. The entry WHO scores were 1-3 in 445 (44%) participants, 4-5 in 529 (52%) participants, and 6 in 38 (4%) participants; and of all participants, 119 died and 271 had disease progression. The odds ratio (OR) associated with COV50 in all 1012 participants for death was 2·44 (95% CI 2·05-2·92) unadjusted and 1·67 (1·34-2·07) when adjusted for sex, age, BMI, comorbidities, and baseline WHO score; and for disease progression, the OR was 1·79 (1·60-2·01) when unadjusted and 1·63 (1·41-1·91) when adjusted (p<0·0001 for all). The predictive accuracy of the optimised COV50 thresholds was 74·4% (71·6-77·1%) for mortality (threshold 0·47) and 67·4% (64·4-70·3%) for disease progression (threshold 0·04). When adjusted for covariables and the baseline WHO score, these thresholds improved AUCs from 0·835 to 0·853 (p=0·033) for death and from 0·697 to 0·730 (p=0·0008) for progression. Of 196 participants who received ambulatory care, 194 (99%) did not reach the 0·04 threshold. The cost reductions associated with 1 day less hospitalisation per 1000 participants were million Euro (M€) 0·887 (5-95% percentile interval 0·730-1·039) in participants at a low risk (COV50 <0·04) and M€2·098 (1·839-2·365) in participants at a high risk (COV50 ≥0·04). INTERPRETATION: The urinary proteomic COV50 marker might be predictive of adverse COVID-19 outcomes. Even in people with mild-to-moderate PCR-confirmed infections (WHO scores 1-4), the 0·04 COV50 threshold justifies earlier drug treatment, thereby potentially reducing the number of days in hospital and associated costs. FUNDING: German Federal Ministry of Health.

Assessment of Ki-67 proliferation index with deep learning in DCIS (ductal carcinoma in situ).

The proliferation index (PI) is crucial in histopathologic diagnostics, in particular tumors. It is calculated based on Ki-67 protein expression by immunohistochemistry. PI is routinely evaluated by a visual assessment of the sample by a pathologist. However, this approach is far from ideal due to its poor intra- and interobserver variability and time-consuming. These factors force the community to seek out more precise solutions. Virtual pathology as being increasingly popular in diagnostics, armed with artificial intelligence, may potentially address this issue. The proposed solution calculates the Ki-67 proliferation index by utilizing a deep learning model and fuzzy-set interpretations for hot-spots detection. The obtained region-of-interest is then used to segment relevant cells via classical methods of image processing. The index value is approximated by relating the total surface area occupied by immunopositive cells to the total surface area of relevant cells. The achieved results are compared to the manual calculation of the Ki-67 index made by a domain expert. To increase results reliability, we trained several models in a threefold manner and compared the impact of different hyper-parameters. Our best-proposed method estimates PI with 0.024 mean absolute error, which gives a significant advantage over the current state-of-the-art solution.

COVID-19 Diagnostics Outside and Inside the National Health Service: A Single Institutional Experience.

The COVID-19 epidemic has been going on continuously for more than 1.5 years. Fast and reliable diagnosis is a key component of an outbreak response strategy. Our goal is to present the statistics from one of the diagnostic points of a large city in Poland. Swabs of the throat or nasopharynx of people reporting for molecular diagnostics of SARS-CoV-2 presence were taken. CE-IVD-certified RNA isolation and RT-PCR assays were used. According to our data, the prevalence of SARS-CoV-2 infection in the examined population equaled 14.7%; however, large differences were observed depending on where the sampling point was located: as much as 50.3% of positive results for samples collected at a stationary point, 36.2% for samples from inpatients and hospital staff, and only 8.9% for samples from patients whose test was paid by their employer. The age structure of the infected population was fairly even, with a slightly higher number of people over 50 years of age. Men were examined more often, but it was among women that a higher percentage of infection was recorded. Every fifth test was performed for a foreigner, but compared to Poles, a much lower incidence of infection was found in these samples. We conclude that due to the high prevalence of infection in patients from social care centers and in those referred to hospitals, it is recommended that a special sanitary regime is followed in those settings. We will evaluate the effectiveness of vaccinations, expecting that the coming months bring positive changes in the statistics on prevalence.

Ki-67 evaluation in breast cancer: The daily diagnostic practice.

CONTEXT: Breast cancer is the most common malignancy in females. It is routinely classified according to the WHO histological typing. However, there is also a molecular classification of breast cancer which is routinely substituted with surrogate subtypes based on expression of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors and proliferation index (PI). PI is defined as the percentage of Ki-67-positive cells among overall cell population. The method commonly applied by pathologists to determine PI is visual scoring of the sample. Strict recommendations for PI assessment do not exist. Thus, the mode of PI evaluation differs significantly between pathologists. AIMS: The aim of our study was to evaluate the daily approach to defining the PI. SETTINGS AND DESIGN: Four practicing nonscholar pathologists were asked to evaluate PI in cases of invasive breast carcinoma. SUBJECTS AND METHODS: The study was performed on a group of 98 patients diagnosed with invasive breast carcinoma. Immunohistochemical reaction was performed with monoclonal antibody against human Ki-67 antigen using Ventana BenchMark XT. STATISTICAL ANALYSIS USED: Results were compared using Pearson's and Spearman's rank correlation coefficients and Fleiss and Cohen's kappa values. RESULTS: Statistical analysis showed pairwise Pearson's coefficients ranging between 0.77 and 0.84 (P < 0.001) and Spearman's rank correlation coefficients ranging between 0.68 and 0.83 (P < 0.001). The Fleiss kappa value for the 14% cutoff point was 0.58 whereas for the 20% cutoff point was 0.60. The pairwise Cohen's kappa values ranged from 0.45 to 0.69 for the 14% cutoff point and 0.53 to 0.67 for the 20% cutoff point. Friedman's rank ANOVA test showed significant differences among the four pathologists (P < 0.001). CONCLUSIONS: Our study shows a significant difference in results and methods of evaluation of PI between pathologists.

Proliferation Index Evaluation in Breast Cancer Using ImageJ and ImmunoRatio Applications.

BACKGROUND: Proliferation index (PI) plays a critical role in distinguishing surrogate biological subtypes of breast cancer and, thus, determining the optimal therapeutic scheme. The commonly applied method of determining PI is visual semiquantitative scoring. The most precise way of PI evaluation is formal counting of nuclei on a digital picture using software equipped with a pointer function. The less time-consuming solution may be using image analysis software enabling automatic counting of nuclei, such as the free web application ImmunoRatio. MATERIALS AND METHODS: We analyzed a group of 98 patients diagnosed with invasive breast carcinoma. A digital image of the hot-spot was taken from each case. Cell Counter plug-in of ImageJ platform was employed to precisely count brown- and blue-stained nuclei. The same images were analyzed using ImmunoRatio. The results were compared using Pearson's and Spearman's coefficients. The agreement was assessed with Cohen's kappa. RESULTS: Pearson's correlation coefficient was 0.84 (p<0.05), Spearman's correlation coefficient was 0.83 (p<0.05). Moderate agreement was shown by Cohen's kappa calculation (K= 0.47; p<0.05). CONCLUSION: As many as 26 cases were classified to different biological subtypes depending on the method of PI assessment. Thus, ImmunoRatio needs further improvement to be a reliable diagnostic tool.

[Yamanaka's factors and core transcription factors--the molecular link between embryogenesis and carcinogenesis]. / Czynniki Yamanaki i rdzeniowe czynniki transkrypcyjne--molekularne ogniwa miedzy embriogeneza i karcynogeneza.

Oct4 and Sox2 transcription factors (belonging to the Yamanaka's factor family) and Nanog, named together as core transcription factors of pluripotency, are indispensable to induce and maintain the pluripotency state. They act generally as activators of genes coding for transcription factors, cofactors and chromatin regulators. They also activate microRNA expression. In addition, Oct4, Sox2 and Nanog function as repressors of genes for factors responsible for escape from pluripotency and differentiation. Core transcription factors positively regulate their own promoters, forming a positive-feedback loop. In recent times, researchers' attention has been attracted towards Oct4, Sox2 and Nanog as potential markers for cancer stem cells (CSCs). The expression of these factors has been confirmed in numerous types of tumors. The aim of this paper is to concisely review features of core transcription factors and their role in embryogenesis and tumorigenesis including the CSC hypothesis.

Adrenal cortical carcinoma with extension into the inferior vena cava--case report and literature review.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with an incidence rate of 1 to 2 per million person-years. ACC most commonly arises sporadically, but may be associated with familial tumour syndromes. Clinical symptoms are mainly related to an excess of steroid hormones. We present an unusual case of adrenocortical carcinoma in a 27-year-old male who complained of non specific mass-effect related symptoms of slowly growing intensity differing from others described in literature because of the patient's age and the sudden deterioration of the clinical course. The tumour was resected with the left kidney with an extension into the inferior vena cava. Histological examination revealed morphological features characteristic of an adrenal cortical tumour. The immunohistochemical results (positive reactions for vimentin, CD56, inhibin, melan A, synaptophysin, bcl-2, calretinin) confirmed the diagnosis. According to the most widely used modified Weiss criteria and the Van Slooten system, a diagnosis of adrenal cortical carcinoma was strongly confirmed. The postoperative condition was poor. Reoperation was conducted, including abdominal aorta thrombectomy and aortic prosthesis implantation. The patient died two days after the second operation. Autopsy revealed a metastatic tumour in the left lung and morphological symptoms of acute circulatory collapse due to a massive haemorrhage into the abdominal cavity, which was the direct cause of death. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1602226377106882.

Cancer stem cells--the current status of an old concept: literature review and clinical approaches.

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.

Cancer stem cells - the current status of an old concept: literature review and clinical approaches

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.

Massage-induced morphological changes of dense connective tissue in rat's tendon.

The aim of the experiment was to determine if possible changes in connective tissue induced by massage could have a positive effect justifing the use of massage in all post-traumatic connective tissue conditions, e.g. tendon injuries. The investigations were performed in a group of 18 Buffalo rats. The rats were divided into two groups (experimental and control). To standardize the massage procedure, it was performed with an algometer probe of 0.5 cm2 with constant pressure force of 1 kG (9,81 N). To analyse the number and diameter of collagen fibrils, two electron micrographs were performed for each rat of the collected segments of tendons of rat tail lateral extensor muscle. After image digitalization and calibration, the measurements were carried out using iTEM 5.0 software. The number of fibrils, their diameter and area were measured in a cross-sectional area. An increase of the number of collagen fibrils was observed in the tendons of massaged animals compared to the control group. Our study demonstrated that massage may cause a beneficial effect on metabolic activity of tendon's fibroblasts and, in consequence, may be applied for more effective use of massage for the prevention of tendon injury as well as after the injury has occurred. (Folia Histochemica et Cytobiologica 2013, Vol. 51, No. 1, 103-106).