The study aimed to compare the time efficiency of manual versus semi-automated FISH spot counting. Three trials each were conducted for manual counting, semi-automated counting, and a combined method. Manual counting took an average of 8.7 minutes per trial while semi-automated counting took an average of 24 minutes per trial. Semi-automated counting also showed a statistically significant error rate of 11.9% compared to manual counting benchmarks. Combining both methods reduced the error rate to only 0.1% while taking an average of 18.6 minutes per trial, suggesting the combined method may provide the best balance of accuracy and efficiency.
In Vivo Bioluminescent / Fluorescent Imagingachang07
The presentation is an introduction to using bioluminescent "reagents" to evaluate drug efficacy in tumor models. This presentation briefly highlights one of many research platforms available at Caliper Life Sciences\' Discovery Alliances\' In Vivo Division that performs contract research for the life science community.
Automated Diagnosis of Malaria in Tropical Areas Using 40X Microscopic Images...CSCJournals
This paper proposes a new algorithm to measure parasitemia stemming from Plasmodium
falciparum by using optical images of capillary blood smears from Sub-Saharan Africa. The
approach is an improvement of the previous noteworthy method by developing a two-tone
adaptive median filter and Sauvola segmentation. The analysis was performed on the database
of 100X and 40X microscopic images originating from real time collection of patients’ blood of
some Cameroon’s laboratories. The obtained results were very satisfactory with a detection of
infected cells on 40X images of a sensitivity at 81.58%, a specificity at 97.11% and an accuracy
at 96.71%. Compared to the previous works, the values portray a real improvement in most
performance’s criteria for 100X images and 40X images as well. There is a significant step of
automated Malaria diagnosis in tropical areas and in the performance assessed. The results of
the method gave more strength for the two magnification ranges. The time spent in analysis is
obviously reduced with 40X magnification and the inaccessibility of the information by the visual
laboratory measurement is henceforth genuinely assaulted. The low cost of the system opens
therefore the possibility of a cost effective method of diagnosing malaria in low and middleincome
countries
Picturing Science: An overview of Imaging TechnologiesDr. Sima Salahshor
In the past decades imaging technologies are increasingly used to model the dynamics and structure of biological systems. Biomedical imaging is now an integral part of biological and medical sciences and is used in both clinical practice and research. In this session some of the latest imaging technologies were reviewed.
In Vivo Bioluminescent / Fluorescent Imagingachang07
The presentation is an introduction to using bioluminescent "reagents" to evaluate drug efficacy in tumor models. This presentation briefly highlights one of many research platforms available at Caliper Life Sciences\' Discovery Alliances\' In Vivo Division that performs contract research for the life science community.
Automated Diagnosis of Malaria in Tropical Areas Using 40X Microscopic Images...CSCJournals
This paper proposes a new algorithm to measure parasitemia stemming from Plasmodium
falciparum by using optical images of capillary blood smears from Sub-Saharan Africa. The
approach is an improvement of the previous noteworthy method by developing a two-tone
adaptive median filter and Sauvola segmentation. The analysis was performed on the database
of 100X and 40X microscopic images originating from real time collection of patients’ blood of
some Cameroon’s laboratories. The obtained results were very satisfactory with a detection of
infected cells on 40X images of a sensitivity at 81.58%, a specificity at 97.11% and an accuracy
at 96.71%. Compared to the previous works, the values portray a real improvement in most
performance’s criteria for 100X images and 40X images as well. There is a significant step of
automated Malaria diagnosis in tropical areas and in the performance assessed. The results of
the method gave more strength for the two magnification ranges. The time spent in analysis is
obviously reduced with 40X magnification and the inaccessibility of the information by the visual
laboratory measurement is henceforth genuinely assaulted. The low cost of the system opens
therefore the possibility of a cost effective method of diagnosing malaria in low and middleincome
countries
Picturing Science: An overview of Imaging TechnologiesDr. Sima Salahshor
In the past decades imaging technologies are increasingly used to model the dynamics and structure of biological systems. Biomedical imaging is now an integral part of biological and medical sciences and is used in both clinical practice and research. In this session some of the latest imaging technologies were reviewed.
Toxicological Screening and Quantitation Using Liquid Chromatography/Time-of-...Annex Publishers
In recent years, an increasing number of new designer-drugs have increased the demands for general toxicological screening [1]. Limited screening based on immunoassays is commonly used in clinical toxicology, whereas more comprehensive approaches are common in forensic toxicology such as screening based on Gas or Liquid Chromatography (GC or LC) approaches [2]. The classic approach has been gas chromatography-Mass Spectrometry (GC-MS) combined with LC-diode-array detection (DAD) for systematic toxicological analysis. This setup has the advantage of covering a very broad spectrum of drugs and illicit substances when combined with library search facilities. However, the analytical sensitivity and specificity of LC-DAD may not be optimal. Thus, more sensitive and specific screening techniques based exclusively on LC combined with mass spectrometry have gained popularity. Multi-target screening and quantitation methods based on LC-tandem mass spectrometry (MS/MS) may provide detection of hundred or more compounds [3]. Using ion-trap MSn detection, several hundred compounds can be detected [4]. A more extended screening is possible using time-of-flight (TOF) mass spectrometry, which is a high-resolution mass spectrometry technique that detects drugs on the basis of their exact mass. Using this technique, scanning is performed over all masses for low molecular drugs, and detected signals can be related to a library of exact drug masses. Retention time and fragmentation pattern contribute to the identification. In principle, it is possible to screen for thousands of compounds, although issues related to software capabilities may limit the number of compounds to several hundred in daily practice [5-7].
Nanotechnology and its Application in Cancer TreatmentHasnat Tariq
Nanotechnology
Nanomaterials
Nanostructures
Nanoparticles
Unexpected Optical Properties of Nanoparticles
Synthesis of Nanoparticles
Nanotechnology in Cancer Treatment
Role of Sulfur NPs in Cancer Treatment
Human Tumour Cell Lines Used in Research
Ehrlich ascites carcinoma (EAC)
Sulfur Nanoparticles Preparation
MTT Assay
Sulphorhodamine-B (SRB) Assay
Median lethal dose (LD 50)
Experimental design
FT-IR Characterization of Sulfur Nanoparticles
SEM Characterization of Sulfur Nanoparticles
EDS Characterization of Sulfur Nanoparticles
XRD Characterization of Sulfur Nanoparticles
Chemical Studies on Sulfur Nanoparticles In Vitro
Biochemical investigations
Conclusion
Applications of Nanoparticles in cancer treatment
Nanoshells
Nano X-Ray therapy
Drug Delivery by Nanoparticles
A novel algorithm for detection of tuberculosis bacilli in sputum smear fluor...IJECEIAES
This work proposes an algorithm aimed at recognizing and accounting Koch bacilli in digital images of microbiological sputum samples stained with auramine, in order to determine the degree of concentration and the state of the disease (tuberculosis). The algorithm was developed with the main objective of maximizing the sensitivity and specificity of the analysis of microbiological samples (recognition and counting of bacilli) according to each preparation method (direct and diluted pellets) in order to reduce the subjectivity of the visual inspection applied by the specialist at the time of analyzing the samples. The proposed algorithm consists of a background removal, an image improvement stage based on consecutive morphological closing operations, a segmentation stage of objects of interest based on thresholdization and a classification stage based on SVM. Each algorithmic stage was developed taking into account the method of preparation of the sample to be processed, being this aspect the main contribution of the proposed work, since it was possible to achieve very satisfactory results in terms of specificity and sensitivity. In this context, sensitivity levels of 91.24% and 93.79% were obtained. Specificity levels of 90.33% and 94.85% were also achieved for direct and diluted pellet methods respectively.
Crimson Publishers -A Sensor Multiplatform for Non Invasive Diagnosis of Pros...CrimsonPublishers-SBB
Crimson Publishers -A Sensor Multiplatform for Non Invasive Diagnosis of Prostate Cancer By A D'Amico in Significances of Bioengineering & Biosciences
This work underlines the utility of a multiplatform based on the use of both artificial lactation and taste systems that show the ability to perform both gas and bio liquid chemical imaging. Preliminary results related to the investigation of prostate cancer suggest that high performance levels with diagnostic and screening purposes can be reached with non invasive experimental procedures.
Nanotechnology essentially restructures molecules to make materials lighter, stronger, more penetrating or absorbant, among many innovative qualities. In cancer research, it offers a unique opportunity to study and interact with normal and cancer cells in real time, at the molecular and cellular scales, and during the various stages of the cancer process. For cancer researchers, a special interest lies in ligand-targeted therapeutic nanoparticles (TNP), which are expected to selectively deliver drugs and especially cytotoxic agents specifically to tumor cells and enhance intracellular drug accumulation. Targeting can be achieved by various mechanisms. For example, nanoparticles with numerous targeting ligands can provide multi-valent binding to the surface of tumor cells with high receptor density (as opposed to low receptor density on normal cells) or nanoparticle agents can enhance permeability and retention (EPR) effect to exit blood vessels in the tumor, to target surface receptors on tumor cells, and to enter tumor cells by endocytosis before releasing their drug payloads.
In this presentation we shall look at nanotechnology in drug development with a focus on anticancers and the advantages of nanoparticles as therapeutic platform technology. Approved nanotech based drugs and their clinical trials will be discussed. Two specific clinical trial case studies will be focused on along at some length with a mention of some ongoing clinical trials of nanotherapeutics. We shall also take a look at the future direction of nanotechnology based therapeutics.
Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.
Toxicological Screening and Quantitation Using Liquid Chromatography/Time-of-...Annex Publishers
In recent years, an increasing number of new designer-drugs have increased the demands for general toxicological screening [1]. Limited screening based on immunoassays is commonly used in clinical toxicology, whereas more comprehensive approaches are common in forensic toxicology such as screening based on Gas or Liquid Chromatography (GC or LC) approaches [2]. The classic approach has been gas chromatography-Mass Spectrometry (GC-MS) combined with LC-diode-array detection (DAD) for systematic toxicological analysis. This setup has the advantage of covering a very broad spectrum of drugs and illicit substances when combined with library search facilities. However, the analytical sensitivity and specificity of LC-DAD may not be optimal. Thus, more sensitive and specific screening techniques based exclusively on LC combined with mass spectrometry have gained popularity. Multi-target screening and quantitation methods based on LC-tandem mass spectrometry (MS/MS) may provide detection of hundred or more compounds [3]. Using ion-trap MSn detection, several hundred compounds can be detected [4]. A more extended screening is possible using time-of-flight (TOF) mass spectrometry, which is a high-resolution mass spectrometry technique that detects drugs on the basis of their exact mass. Using this technique, scanning is performed over all masses for low molecular drugs, and detected signals can be related to a library of exact drug masses. Retention time and fragmentation pattern contribute to the identification. In principle, it is possible to screen for thousands of compounds, although issues related to software capabilities may limit the number of compounds to several hundred in daily practice [5-7].
Nanotechnology and its Application in Cancer TreatmentHasnat Tariq
Nanotechnology
Nanomaterials
Nanostructures
Nanoparticles
Unexpected Optical Properties of Nanoparticles
Synthesis of Nanoparticles
Nanotechnology in Cancer Treatment
Role of Sulfur NPs in Cancer Treatment
Human Tumour Cell Lines Used in Research
Ehrlich ascites carcinoma (EAC)
Sulfur Nanoparticles Preparation
MTT Assay
Sulphorhodamine-B (SRB) Assay
Median lethal dose (LD 50)
Experimental design
FT-IR Characterization of Sulfur Nanoparticles
SEM Characterization of Sulfur Nanoparticles
EDS Characterization of Sulfur Nanoparticles
XRD Characterization of Sulfur Nanoparticles
Chemical Studies on Sulfur Nanoparticles In Vitro
Biochemical investigations
Conclusion
Applications of Nanoparticles in cancer treatment
Nanoshells
Nano X-Ray therapy
Drug Delivery by Nanoparticles
A novel algorithm for detection of tuberculosis bacilli in sputum smear fluor...IJECEIAES
This work proposes an algorithm aimed at recognizing and accounting Koch bacilli in digital images of microbiological sputum samples stained with auramine, in order to determine the degree of concentration and the state of the disease (tuberculosis). The algorithm was developed with the main objective of maximizing the sensitivity and specificity of the analysis of microbiological samples (recognition and counting of bacilli) according to each preparation method (direct and diluted pellets) in order to reduce the subjectivity of the visual inspection applied by the specialist at the time of analyzing the samples. The proposed algorithm consists of a background removal, an image improvement stage based on consecutive morphological closing operations, a segmentation stage of objects of interest based on thresholdization and a classification stage based on SVM. Each algorithmic stage was developed taking into account the method of preparation of the sample to be processed, being this aspect the main contribution of the proposed work, since it was possible to achieve very satisfactory results in terms of specificity and sensitivity. In this context, sensitivity levels of 91.24% and 93.79% were obtained. Specificity levels of 90.33% and 94.85% were also achieved for direct and diluted pellet methods respectively.
Crimson Publishers -A Sensor Multiplatform for Non Invasive Diagnosis of Pros...CrimsonPublishers-SBB
Crimson Publishers -A Sensor Multiplatform for Non Invasive Diagnosis of Prostate Cancer By A D'Amico in Significances of Bioengineering & Biosciences
This work underlines the utility of a multiplatform based on the use of both artificial lactation and taste systems that show the ability to perform both gas and bio liquid chemical imaging. Preliminary results related to the investigation of prostate cancer suggest that high performance levels with diagnostic and screening purposes can be reached with non invasive experimental procedures.
Nanotechnology essentially restructures molecules to make materials lighter, stronger, more penetrating or absorbant, among many innovative qualities. In cancer research, it offers a unique opportunity to study and interact with normal and cancer cells in real time, at the molecular and cellular scales, and during the various stages of the cancer process. For cancer researchers, a special interest lies in ligand-targeted therapeutic nanoparticles (TNP), which are expected to selectively deliver drugs and especially cytotoxic agents specifically to tumor cells and enhance intracellular drug accumulation. Targeting can be achieved by various mechanisms. For example, nanoparticles with numerous targeting ligands can provide multi-valent binding to the surface of tumor cells with high receptor density (as opposed to low receptor density on normal cells) or nanoparticle agents can enhance permeability and retention (EPR) effect to exit blood vessels in the tumor, to target surface receptors on tumor cells, and to enter tumor cells by endocytosis before releasing their drug payloads.
In this presentation we shall look at nanotechnology in drug development with a focus on anticancers and the advantages of nanoparticles as therapeutic platform technology. Approved nanotech based drugs and their clinical trials will be discussed. Two specific clinical trial case studies will be focused on along at some length with a mention of some ongoing clinical trials of nanotherapeutics. We shall also take a look at the future direction of nanotechnology based therapeutics.
Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.
In situ hybridization methods and techniques course slides Pat Heslop-HarrisonPat (JS) Heslop-Harrison
Methods and techniques for chromosomal in situ hybridization and molecular cytogenetics. Fixations, chromosomes preparation, mostly using plant chromosomes, hybridiziation mixtures, stringency calculations and fluorescent microscopy.Trude Schwarzacher and Pat Heslop-Harrison
A novel convolutional neural network based dysphonic voice detection algorit...IJECEIAES
This paper presents a convolutional neural network (CNN) based noninvasive pathological voice detection algorithm using signal processing approach. The proposed algorithm extracts an acoustic feature, called chromagram, from voice samples and applies this feature to the input of a CNN for classification. The main advantage of chromagram is that it can mimic the way humans perceive pitch in sounds and hence can be considered useful to detect dysphonic voices, as the pitch in the generated sounds varies depending on the pathological conditions. The simulation results show that classification accuracy of 85% can be achieved with the chromagram. A comparison of the performances for the proposed algorithm with those of other related works is also presented.
Professor Harrison Bai, Artificial Intelligence Applications in Radiology_mHe...Levi Shapiro
Artificial Intelligence Applications in Radiology, presentation by Dr Harrison Bai, Assistant Professor of Diagnostic Imaging, Warren Alpert Medical School, Brown University. His research interests focus on AI, machine learning, and computer vision as applied to medical image analysis. Dr Bai is an associate editor for the journal Radiology: Artificial Intelligence and is currently a principal investigator for an RSNA Research Scholar grant and an NIH grant. The AI Radiology Lab has various areas of work including COVID-19; Treatment response assessment on imaging (brain, TACE, lung, colorectal); Rapid diagnosis of large-vessel ischemic stroke, patient selection and outcome prediction; Tumor characterization on imaging; Infrastructure development; Federated learning; Image registration (CT-guided tumor ablation); Radiology reports natural language processing. The AI pipeline includes DIANA system, Diagnosis model, severity model and progression model across various automated features and the value proposition. One Technique for dealing with missing sequence and imaging artifact- Sequence dropout. Human-in-the-loop AI. In the short- to mid-term, the utilization of AI needs to be combined with human intervention and supervision. Active learning strategy – annotation. Treatment response evaluation on imaging. Automatic quality estimation to flag the failed cases for humans to review and/or edit. Human in the loop annotation. Automatic quality estimation. Federated learning. Semi-supervised and unsupervised learning. AWS NVIDIA Clara Train SDK using TensorFlow 1.14. Annotations vary across imaging sites. Share weights without sharing data. Domain shift – distribution difference between source data and target data leading to performance degradation.
In this paper we present the use of a signal processing technique to find dominant channels in
near infrared spectroscopy (NIRS). Cross correlation is computed to compare measuring
channels and identify delays among the channels. In addition, visual inspection was used to
detect potential dominant channels. The results showed that the visual analysis exposed painrelated
activations in the primary somatosensory cortex (S1) after stimulation which is
consistent with similar studies and the cross correlation analysis found dominant channels on
both cerebral hemispheres. The analysis also showed a relationship between dominant channels
and neighbouring channels. Therefore, our results present a new method to detect dominant
regions in the cerebral cortex using near-infrared spectroscopy. These results have also
implications in the reduction of number of channels by eliminating irrelevant channels for the
experiment.
CROSS CORRELATION ANALYSIS OF MULTI-CHANNEL NEAR INFRARED SPECTROSCOPYcscpconf
In this paper we present the use of a signal processing technique to find dominant channels in near infrared spectroscopy (NIRS). Cross correlation is computed to compare measuring channels and identify delays among the channels. In addition, visual inspection was used to detect potential dominant channels. The results showed that the visual analysis exposed painrelated activations in the primary somatosensory cortex (S1) after stimulation which is consistent with similar studies and the cross correlation analysis found dominant channels on both cerebral hemispheres. The analysis also showed a relationship between dominant channels
and neighbouring channels. Therefore, our results present a new method to detect dominant regions in the cerebral cortex using near-infrared spectroscopy. These results have also implications in the reduction of number of channels by eliminating irrelevant channels for the experiment.
CROSS CORRELATION ANALYSIS OF MULTI-CHANNEL NEAR INFRARED SPECTROSCOPYcscpconf
In this paper we present the use of a signal processing technique to find dominant channels in near infrared spectroscopy (NIRS). Cross correlation is computed to compare measuring channels and identify delays among the channels. In addition, visual inspection was used to detect potential dominant channels. The results showed that the visual analysis exposed painrelated activations in the primary somatosensory cortex (S1) after stimulation which is consistent with similar studies and the cross correlation analysis found dominant channels on both cerebral hemispheres. The analysis also showed a relationship between dominant channels and neighbouring channels. Therefore, our results present a new method to detect dominant regions in the cerebral cortex using near-infrared spectroscopy. These results have also implications in the reduction of number of channels by eliminating irrelevant channels for the experiment
ijerst offers a fast publication schedule whilst maintaining rigorous peer review; the use of recommended electronic formats for article delivery expedites the process.
International Journal of Engineering Research and Science & Technology (IJERST) is an international online journal in English published Quarterly. All submitted research articles are subjected to immediate rapid screening by the editors, in consultation with the Editorial Board or others working in the field as appropriate, to ensure they are likely to be of the level of interest and importance appropriate for the journal.
ijerst offers a fast publication schedule whilst maintaining rigorous peer review; the use of recommended electronic formats for article delivery expedites the process. Editorial Board or others working in the field as appropriate, to ensure they are likely to be of the level of interest and importance appropriate for the journal. International Journal of Engineering Research and Science & Technology (IJERST) is an international online journal in English published Quarterly. All submitted research articles are subjected to immediate rapid screening by the editors.
The Karolinska Institute (KI) is the largest centre for medical education and research in Sweden and the home of the Nobel Prize in Physiology or Medicine.
KI consists of 22 departments and 600 research groups dedicated to improving human health through research and higher education.
The role of the Kohonen/Grafström team has been to guide the application, analysis, interpretation and storage of so called “omics” technology-derived data within the service-oriented subproject “ToxBank”.
Data analytics to support exposome research course slidesChirag Patel
We present new publicly available tools to bootstrap your own data-driven investigations to correlate the environment with phenotype. Course materials here: http://www.chiragjpgroup.org/exposome-analytics-course/
Developing a framework for for detection of low frequency somatic genetic alt...Ronak Shah
Cancer is a complex, heterogeneous disease of the genome. Most cancers result
from an accumulation of multiple genetic alterations that lead to dysfunction of cancer-associated
genes and pathways. Recent advances in sequencing technology have enabled comprehensive
profiling of genetic alterations in cancer. We have established a targeted sequencing platform
(IMPACT: Integrated Mutation Profiling of Actionable Cancer Targets) using hybridization capture and
next-generation sequencing (NGS) technology, which can reveal mutations, indels and copy number
alterations involving 340 cancer related genes.
1. Introduction
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that
uses fluorescently labeled probes to detect chromosomal
abnormalities. In situ hybridization was first used in the 1960s, and the
use of fluorescent probes quickly followed (Rudkin and Stollar, 1977).
A fluorescently labeled probe is designed to attach to a particular area
of a chromosome. Its presence then signifies the presence of the gene
located in that area, appearing as colored dots among the also
fluorescently stained cell background (Carothers, 1994). Trained
technologists can detect chromosomal abnormalities like
translocations, inversions, duplications and deletions. Unlike
conventional cytogenetic analysis, results can be obtained quickly,
leading to less parental anxiety in the case of prenatal samples and
speeding up diagnosis and treatment. With the widespread adoption
and popularization of the FISH, products have emerged to aide this
technique. Namely, automated spot counting software has been
developed by a number of manufacturers in an effort to assist
technologists and increase efficiency (Vrolijk, 1996; Zhou, 2007). Much
of the literature searched related to FISH spot counting systems were
published in the early to mid-2000s during the peak of its flourish (van
der Logt, 2015). The automated BioView Duet counting system was
compared to typical manual scoring using a BCR/ABL dual-fusion
probe on chronic myeloid leukemia (CML) specimens to evaluate for
minimal residual disease. It has been concluded that the Duet system
appears to be more sensitive and cost-effective than manual scoring
for CML FISH specimens (Knudson, 2007). The aim of this study is to
determine whether a semi-automatic FISH spot-counting system will
be more time-efficient than manual spot-counting. The hypothesis for
this study is that manual spot-counting will prove more time-efficient.
Efficiency and turnaround times are important metrics for laboratory,
as faster diagnosis could lead to faster treatment or a wider array of
decisions. Moreover, financially determining efficiency could lead to
allocation of resources in different areas. Fully automatic spot-
counting systems are a significant monetary investment, but semi-
automatic systems may be a viable, cost-saving alternative to fully
automatic systems if they prove more efficient than manual
technologist scoring.
Discussion
The study was designed into three major parts with three trials for the
first two parts and two trials for the last part. The first section, semi-
automated FISH counting, relied solely on the system for signal analysis
with minor interactive correction as needed. The second section was
conducted solely using manual counting (Ravkin, 1999). The last part of
the study combined both methods (conjugated method) to evaluate the
possibility of obtaining an optimal result. A total of 314 nuclei were
scanned and analyzed for the first two parts of the experiment, and
another 200 nuclei were scored for the last section. The hypotheses of
the study was that the semi-automated FISH counting technique is more
efficient and the conventional FISH counting technique is superior in
terms of accuracy.
As seen in Tables 1, there was a significant deviation in the semi-
automated counts. Using manual counts as the benchmark, 73.2% of the
signal patterns were 2R0G. Semi-automated counts for 2R0G for the
same slide were 61.3%, a difference of 11.9%. This is 6.9% above the
standard cutoff of 5%, and thus a statistically significant error. This error
is attributed to a technical limitation of the system when reading
Spectrum Orange signals; CCD camera sensitivity and uncorrected
autofluorescence are known culprits. Another likely source for errors is
poor probe quality, a result of non-ideal hybridization efficiency (De
Solórzano, 1998). This causes high background noise or weak signals.
Misclassification of artefactual organic residues - often called debris –
can be misinterpreted as FISH signals as well (Netten, 1997). Systematic
deviations such as overlapping signals, signals with irregular size and
shape (due to different condensation states), or out-of-focus images
contribute to error as well. All of these may occur if they were not
completely removed in the earlier exclusion step. Less than 5%
disagreement between semi-automated and manual scoring was
achieved for the rest of signal patterns.
The manual FISH counts determine the benchmark for accuracy,
presuming that the error due to the technician is minimized. Running
manual counts in triplicate and with different technicians minimizes this
error. Manual counts were obtained more quickly than with the semi-
automated system (26 min vs. 72 min) (Table 1). However, this result may
be misleading since 45 minutes were required for the technician to
become familiarized with the system. Adjusting for this difference makes
the times about the same, but this can change depending on the system
and the technician. Further experiment is needed in order to verify the
efficiency of the semi-automated counting result. When combining both
methods, the accuracy of the quantitated signals was significantly
increased, resulting in only a 0.1% difference for the 2R0G signal pattern
– down from the initial disagreement of 11.9%.
Carothers, A. D. (1994). Counting, measuring, and mapping in fish‐labelled cells: Sample size
considerations and implications for automation. Cytometry, 16(4), 298-304.
De Solórzano, C. O., Santos, A., Vallcorba, I., Garcia-Sagredo, J. M., & del Pozo, F. (1998).
Automated FISH spot counting in interphase nuclei: Statistical validation and data correction.
Cytometry, 31(2), 93-99.
Knudson, R., Shearer, B., & Ketterling, R. (2007). Automated Duet spot counting system and manual
technologist scoring using dual-fusion fluorescence in situ hybridization (D-FISH) strategy:
Comparison and application to FISH minimal residual disease testing in patients with chronic myeloid
leukemia. Cancer Genetics and Cytogenetics, 175(1), 8-18.
Netten, H., Young, I. T., van Vliet, L. J., Tanke, H. J., Vroljik, H., & Sloos, W. C. (1997). FISH and
chips: automation of fluorescent dot counting in interphase cell nuclei. Cytometry, 28(1), 110.
Prins, M. J. D., Ruurda, J. P., van Diest, P. J., van Hillegersberg, R., & ten Kate, F. J. W. (2013).
Evaluation of the HER2 amplification status in oesophageal adenocarcinoma by conventional and
automated FISH: a tissue microarray study. Journal of clinical pathology, jclinpath-2013.
Ravkin, I., & Temov, V. (1999, June). Automatic counting of FISH spots in interphase cells for
prenatal characterization of aneuploidies. In BiOS'99 International Biomedical Optics Symposium (pp.
208-217). International Society for Optics and Photonics.
Rudkin, G. T. and Stollar, B. D. (1977). High resolution detection of DNA RNA hybrids in situ by
indirect immunofluorescence. Nature 265,472 -473.
van der Logt, E. M., Kuperus, D. A., van Setten, J. W., van den Heuvel, M. C., Boers, J. E., Schuuring,
E., & Kibbelaar, R. E. (2015). Fully Automated Fluorescent in situ Hybridization (FISH) Staining and
Digital Analysis of HER2 in Breast Cancer: A Validation Study. PloS one, 10(4), e0123201.
Vrolijk, H., Sloos, W. C., van de Rijke, F. M., Mesker, W. E., Netten, H., Young, I. T., ... & Tanke, H.
J. (1996). Automation of spot counting in interphase cytogenetics using brightfield microscopy.
Cytometry, 24 (2), 1996, p. 158-166.
Zhou, Z., Pons, M. N., Raskin, L., & Zilles, J. L. (2007). Automated image analysis for quantitative
fluorescence in situ hybridization with environmental samples. Applied and environmental
microbiology, 73(9), 2956-2962.
Materials and Methods
The system used for FISH counting consists of: a fluorescence
microscope (Olympus BX51), conductive image capturing system,
processing and analysis software. The major components of the
microscope used in the experiment included a scanning stage, a five
position objective rotor and a five position filter rotor. The automated
FISH counting and analysis was performed using supervised
automated scoring with the CytoVision Version 7.4 digital platform
(Leica Microsystems Inc., Buffalo Grove, IL). The CEPXY-ENG assay
(numerical probe) was used for this study to determine signal patterns,
which were defined as 1R1G, 1R0G, 2R0G, 2R1G, 3R0G before the
image capturing and analysis process (Table 1). The FISH signals were
scored and analyzed through a triple-band filter - DAPI, Spectrum
Orange for the chromosome X probe and Spectrum Green for
chromosome Y probe. The probe separation was set as 1.2 um
(default). The primary objective slides were scanned at 10x
magnification in the DAPI channel of the fluorescence microscope to
check the overall cell distribution on the specimen. Then, a region of
interest (ROI) was selected at 60X magnification and all fluorescent
signals were measured for signal intensity. The red and green spots
were segmented automatically. Background correction was performed
for all the signals obtained from these channels. Captured images
from different focus planes were combined into a composite image.
Based on the density appearance under 60X magnification (average of
20 cells/image), ten images with combined focus planes were captured
in order to obtain and count 314 nuclei in total for automated counting
and analysis. The signals with low intensities in the TRITC and FITC
channel due to nonspecific backgrounds were defined as zero; nuclei
that did not emit any red and green chromatic signals, or the nuclei
with single green signal, along with auto-fluorescing cells were
excluded from the system; overlapping signals or signals with
irregular size and shape due to a different condensation state of the
chromatin or due to the sample preparation were excluded from
selection, so as the cells without clearly defined borders. The time
needed for scanning each region of interest was recorded and the total
time period used for the entire process was recorded post experiment.
Results References
The same signal patterns were applied to all trials: 1R1G, 2R0G, 1R0G,
2R1G, and 3R0G. In trial 1, a total of 132 nuclei were scored, with a
result of 28% 1R1G, 61.4% 2R0G, and 10.6% 1R0G signal patterns. An
amount of 103 cells were selected for trial 2, and 18.4% 1R1G, 63.1%
2R0G, 18.4% 1R0G were obtained. With a total of 79 cells chosen,
31.6% showed 1R1G, 59.5% showed 2R0G, and 8.9% showed 1R0G.
The average percentages of different signal patterns for three trials
calculated were: 26% 1R1G, 61.3% 2R0G, and 12.6% 1R0G. The time
used for each trial were recorded to be 45 minutes, 20 minutes and 7
minutes respectively, and the total time spent on semi-automated
FISH counting was 72 minutes (Table 1). The same 314 nuclei were
used for manual scoring. The percentages for signal patterns of 1R1G,
2R0G, and 1R0G in trial 1 were 29.5%, 68.9%, and 1.5% respectively,
21.4%, 78.6%, 0 in trial 2, and 26.6%, 72.2%, 1.3% in trial 3. An average
of 25.8%, 73.2%, and 0.93% for signal patterns of 1R1G, 2R0G and
1R0G were generated respectively. The time used for the three trials
were 12 minutes, 9 minutes, and 5 minutes, totaling 26 minutes for the
manual FISH counting process.
The accuracy of semi-automated FISH counting was determined by
measuring the difference observed from trials that had been verified
manually. Semi-automated FISH scoring produced the following
ratios: 26% 1R1G, 61.3% 2R0G. Manual FISH scoring showed: 12.6%
1R0G, and 25.8% 1R1G, 73.2% 2R0G, 0.93% 1R0G. The disagreement
between semi-automated and manual counting systems for 1R1G,
2R0G, and 1R0G were 0.2%, 11.9%, and 11.67% respectively.
ISSUES RELEVANT TO FISH SEMI-AUTOMATED SPOT COUNTING SYSTEM
Ken Sterns, Angela Teng, Jiaqi Chen, Cristina Garcia, Maria Valencia, Roberto Guajardo, Dominique Cline, Crystal Lee, Victoria
Nettles, Sylvia Wong, Ming Zhao, and Jun Gu
University of Texas MD Anderson, School of Health Professions
Trials Manual Count: 314 cells (104 cells/trial)
(Internal Control)
Semi-Automated Count: 314 cells
(104cells/trial)
Semi-Automated + Manual: 200 cells
(66 cells/trial)
Signal
Patterns
1R1G 2R0G 1R0G Time
(min)
1R1G 2R0G 1R0G Time
(min)
1R1G 2R0G 1R0G Time
(min)
Trial 1 39(29.5%) 91(68.9%) 2(1.5%) 12 37(28%) 81(61.4%) 14(10.6%) 45 22(31.4%) 47(67.1%) 1(1.4%) 22
Trial 2 22(21.4%) 81(78.6%) 0 9 19(18.4%) 65(63.1%) 19(18.4%) 20 13(20.6%) 49(77.7%) 1(1.6%) 19
Trial 3 21(26.6%) 57(72.2%) 1(1.3%) 5 25(31.6%) 47(59.5%) 7(8.9%) 7 16(23.9%) 50(74.6%) 1(1.5%) 15
Total 82(26.1%) 229(72.9%) 3(0.96%) 26 81(25.8%) 193(61.5%) 40(12.7%) 72 51(25.5%) 146(73%) 3(1.5%) 56
Ave. 27(25.8%) 76(73.2%) 1(0.93%) 8.7 27(26%) 64(61.3%) 13(12.6%) 24 17(25.3%) 48(73.1%) 1(1.5%) 18.6
%diff.
from
Manual
0.2% 11.9% 11.67% 46 min
longer
0.5% 0.1% 0.57% 30 min
longer
Ave. diff. from Manual: 7.9% Ave. diff. from Manual: 0.39%
Figure 1. Captured Images of Semi-
Automated FISH Counting Results under
Fluorescence at 60X. A. Negative control:
CEP X; B. Positive control: CEP Y; C, D, E
are the semi-automated scoring results of
trial 1, trial 2, and trial 3 respectively after
manual corrections.
Table 1. Counting result among different methods.