Advances in Cell and Molecular Diagnostics

Advances in Cell and Molecular Diagnostics

Pongali Raghavendra

REVA University (India) Director of School of Chemical and Biological Sciences, REVA University (India)

Thammineni Pullaiah

Sri Krishnadevaraya University (India)

Table of Contents

Cover image

Title page

Copyright

Preface

Executive Summary

Chapter 1. Cellular and Molecular Diagnostics: An Introduction

1.1. Introduction

1.2. Biological Specimens or Samples

1.3. Cell Phenotype

1.4. Cell-Based Screening Assays

1.5. Cytogenetics-Based Assays

1.6. Cellular Pathways—Diagnostic Target Assays

1.7. Molecular-Based Methods

1.8. Molecular (DNA/RNA) Sample–Based Diagnostics

1.9. Amino Acid

1.10. Protein-Based Diagnostic Assays

Chapter 2. RNA-Based Applications in Diagnostic and Therapeutics for Cancer

2.1. Introduction

2.2. miRNA Working Model and Targets

2.3. miRNAs as Diagnostic Markers

2.4. RNA in Various Cancers

2.5. miRNA-Mediated Pathways in Cancer

2.6. RNA Detection Methods

2.7. RNA as Biomarkers in Cancer Diagnosis

2.8. Therapeutic Applications of RNA

2.9. Conclusion

Chapter 3. Advancements in Genetic Applications for Cellular and Molecular Diagnostics

3.1. Introduction

3.2. Diagnosis of Genetic Diseases

3.3. Genetic Tests

3.4. Prenatal Diagnosis

3.5. Advances in Prenatal Screening

3.6. MicroRNA

3.7. DNA Sequencing and PCR

3.8. Single Nucleotide Polymorphisms (Plasma Placental RNA Allelic Ratio)

3.9. Methylated DNA Precipitation

3.10. Cancer Diagnosis

3.11. Most Recent Techniques in Identifying Chromosomal Aberrations

3.12. DNA Microarrays for Gene Expression Studies

3.13. Analysis of Single-Cell Gene Expression

3.14. Next-Generation Sequencing

3.15. CRISPR and CAS9 in Genome Editing

3.16. Conclusion

Chapter 4. Biomedical Imaging Role in Cellular and Molecular Diagnostics

4.1. Introduction

4.2. Techniques

4.3. Conclusion

Chapter 5. Breast Cancer–Targeted Therapy Using Nanocarriers

5.1. Introduction

5.2. Nanoparticle

5.3. Antibody Drug Conjugates

5.4. Conclusion

Chapter 6. Human Papillomavirus- and Epstein–Barr Virus-Caused Tumor Diagnosis and Therapy

6.1. Introduction

6.2. Human Papillomavirus

6.3. HPV Diagnosis Methods

6.4. HPV Treatment

6.5. Prevention

6.6. Epstein–Barr Virus

6.7. Epstein–Barr Virus (Burkitt Lymphoma) Diagnosis Methods

6.8. Epstein–Barr Virus Treatment

6.9. Conclusion

Chapter 7. Pathogen Identification Using Novel Sequencing Methods

7.1. Introduction

7.2. Importance of Pathogen Detection

7.3. Sequence-Based Virome Analysis

7.4. Microbial Identification

7.5. Antibiotic Resistance and Virulence

7.6. Yeast

7.7. Metagenomics

7.8. 16S Ribosomal Sequencing

7.9. DNA Sequencing

7.10. Next-Generation Sequencing

7.11. Conclusion

Chapter 8. Future of Cellular and Molecular Diagnostics: Bench to Bedside

8.1. Introduction

8.2. Future Aspects—RNA-Based Applications

8.3. Future Aspects—Genetic Applications

8.4. Future Aspects—Biomedical Imaging

8.5. Future Aspects—Nanocarriers

8.6. Future Aspects—HPV/EBV

8.7. Future Aspects—Novel Methods for Pathogen Identification

8.8. Cellular and Molecular Diagnostics—Potentials

8.9. Challenges and Issues

8.10. Final Conclusions

Index

Copyright

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Preface

The book ADVANCES IN CELL AND MOLECULAR DIAGNOSTICS has been prepared keeping in mind the requirements of Graduate and Postgraduate students of Medical Genetics, Biochemistry, Biotechnology, and Microbiology. This book will also be useful to the Diagnostic centres, Medical Practitioners, Pathologists, Researchers, and students of PG diploma and short-term courses in Paramedical and Biomedical sciences. The main thrust of the book is cellular- and molecular-based diagnostics and prognosis.

Starting with an introductory chapter, the book covers all the features of fundamental understanding of the conceptual cellular and molecular level areas and further advance to honing the end user skills, staying abreast with the recent advancements. Major effort has been paid to make the material comprehensive, concise, and practical.

The concepts in the book have been presented covering both theoretical knowledge, which is crucial, and practical applications for the holistic development of a student and researcher. The contents are covered and balanced for both theoretical and practical real-life clinical applications.

The fast growing technologies and variety and increasing complexity of cellular or molecular testing methodologies are well described using a larger clinically relevant–based examples. Often there is a disconnect between the advancing edge of the practice of individualized/personalized medicine and diagnostic, prognostic, and/or therapeutic utility across major diseases. So, this book caters with examples highlighting these gaps and challenges.

Our sincere thanks to Professors, friends, and students who gave constructive feedbacks and suggestions without which this book would not have been in the present form. Special thanks to our critics who helped us to bring out the best. We really respect their opinions.

We express our gratitude to our family members and friends who encouraged us to write this book. We thank Elsevier Editorial team who guided and supported us in bringing out this book.

We keenly look forward to constructive feedback and suggestions from the esteemed readers of the book.

P.B. Raghavendra raghavbiot@gmail.com

T. Pullaiah pullaiah.thammineni@gmail.com

Executive Summary

One of the most important steps in disease biology is early diagnostic, which facilitates to design and choose a better therapeutic approach. Most of the cellular and molecular diagnostic methods serve as tools to understand wide variety of clinically and genetically heterogeneous disorders with specific or overlapping phenotypes. Both the private and public sectors around the globe in recent years have increased investments in field of medical diagnostics for low- or middle-income countries. Despite these investments, numerous barriers prevent the adoption of existing diagnostics or developed new diagnostics in these countries. Appropriate sample collection and identification of biomarker can predict that incidence and severity of disease would allow for improved and targeted therapies to prevent adverse disease outcomes. Identification of specific biomarkers of different disorders might highlight potential clinical opportunities for disease prediction, diagnosis, prognosis, and treatment. Biosensor-based detection techniques have gained a lot of attention with vast applications in medical diagnostics, drug screening, safety, and as well in other fields of health sector. Large-scale automation is enabling sophisticated cellular and molecular tests to be performed in the full scope of health care settings, bringing state of the art diagnostics to all areas of the world. The present book provides an overview of the current landscape for cellular-molecular diagnostics, elaborates the key technologies that are driving the cellular-molecular revolution, illustrates the power of cellular-molecular diagnostics with some specific examples, and concludes by noting several challenges that have the potential to influence progress in the critical field of medicine and health sectors.

Chapter 1

Cellular and Molecular Diagnostics

An Introduction

Abstract

Advances in cell and molecular biology studies have revolutionized the diagnosis and treatment of many different diseases. Cell and molecular diagnostics (CMD) have become an essential part of investigations required in providing optimal care and has become an important tool for the health care providers for better treatment. CMD gives insights into the pathogenesis and prognosis of various diseases. Rapid technological advances have helped to increase the speed and performance of various molecular assays thereby reducing turnaround time, in turn translating to providing better clinical services. With the increase in CMD applications to clinical services, entire world has seen a sudden spurt of cellular and molecular labs. However, this has highlighted an acute shortage of trained laboratory personnel who can carry out cellular or molecular assays in a diagnostic mode. Understanding the mechanisms of disease at the molecular and genetic levels can now be translated into diagnostic, prognostic, and therapeutic applications in modern medicine. Molecules at abnormal levels (quantitative or qualitative) provide not only a signature for the presence of a disease, but also the indication for a drug targeting the specific abnormal function. Concurrently, the role of CMD has expanded from mere study observations to constantly translating new discoveries and novel technologies into useful clinical tests that provide a molecular fingerprint of diseases and that are predictive of the response. In the introductory chapter we will cover the importance of biological sample collection from different methods; cell-based diagnostic methods, cellular pathway diagnostic targets, and molecular-based methods.

Keywords

Cytogenomics; DNA; Fluorescence in-situ hybridization (FISH); RNA; Serum

Chapter Outline

1.1 Introduction

1.2 Biological Specimens or Samples

1.2.1 Lesion Swabs

1.2.2 Saliva

1.2.3 Biopsy

1.2.4 Serum

1.2.5 Plasma

1.2.6 Blood

1.2.7 Cerebrospinal Fluid

1.2.8 Autopsy Tissues

1.3 Cell Phenotype

1.3.1 Cell-Based Diagnostic Methods

1.3.1.1 Fluorescence/Microscopic Assays

1.3.2 Xenodiagnosis

1.3.3 Flow Cytometry

1.4 Cell-Based Screening Assays

1.4.1 Plaque Assay

1.4.2 Tissue Culture Infectious Dose50 Assay

1.5 Cytogenetics-Based Assays

1.5.1 Chromosome Analysis

1.6 Cellular Pathways—Diagnostic Target Assays

1.6.1 WNT/β-catenin Signaling

1.6.2 JAK/STAT Signaling Pathway

1.6.3 Ras-Raf-MEK-ERK-MAPK Pathway

1.7 Molecular-Based Methods

1.7.1 Polymerase Chain Reaction and Real-Time PCR

1.7.2 Fluorescent In Situ Hybridization

1.7.3 DNA Microarrays

1.7.4 Targeted Mutation Analysis

1.7.5 Detection of Unknown Mutations

1.7.6 Detection of Copy Number Variations

1.8 Molecular (DNA/RNA) Sample–Based Diagnostics

1.8.1 Next-Generation Sequencing

1.8.2 MicroRNAs

1.8.2.1 Spinal Cord Injury

1.8.2.2 Bone Diseases

1.8.2.3 Post-Traumatic Stress Disorder

1.8.2.4 Cardiovascular Diseases

1.8.2.5 Diabetes Mellitus

1.8.2.6 HIV/AIDS

1.8.2.7 Forensic Diagnostics

1.9 Amino Acid

1.10 Protein-Based Diagnostic Assays

1.10.1 Enzyme Immunoassay—ELISA

1.10.2 Multiplexed Immunoassays

1.10.3 Microarray-Based Assays

1.10.4 Bead-Based Assay

1.10.5 Proteomic’s Technologies for the Study and Diagnosis of Autoimmune Diseases

References

1.1. Introduction

Cellular and molecular diagnostics is one of the most dynamic and transformative areas of diagnostics, leading to advances in prognosis, research, and treatment that are revolutionizing health care across a wide range of diseases and health conditions. Cellular–Molecular diagnostics is a broad term describing a class of diagnostic tests that assess a person’s health literally at a cellular and molecular level, detecting and measuring specific cellular alterations, genetic sequences in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or amino acids or the proteins they express. Cellular level diagnostics involves the study of the set of cellular parameter modifications in the form of change in cell structural components; chromosomal aberrations; cytogenetic events; cell homeostasis; metabolic, physiological, or phenotype modifications; and relative signaling pathways. Molecular diagnostics identify gene, RNA, and protein variations that shed light on whether a specific person is predisposed to have a disease, whether they actually have a disease, or whether a certain treatment option is likely to be effective for a specific disease. These tests also can detect and quantify the presence of specific viruses, bacteria, or any type of cells. Hence forth after getting insights for Cellular–Molecular diagnostics terminology we will state it as CMD in the rest of our book chapters.

In general human bodily processes, both normal and abnormal, as well as health or disease states, are driven by the interaction of our genes and the proteins they produce that carry out specific functions within the body. Therefore, the ability to quickly and accurately assess an individual’s health at cellular and molecular level is truly transforming the practice of medicine. Specific proteins or genetic sequences have a close association with a specific health condition or disease; they are often referred to as biomarkers because they are markers of that condition or disease.

CMD is a collection of tools that are driving the continuing discovery of biomarkers at the research level, which in turn leads to treatments designed around these biomarkers. CMD plays an additional critical role by ensuring that these new therapies are delivered to the right patients through more accurate diagnosis of the exact nature of individual disease. This has led to the emerging field of companion diagnostics, in which a CMD test is used to identify whether a specific therapy (a companion to the diagnostic) is likely to be effective for an individual patient.

1.2. Biological Specimens or Samples

Measurement of biomarkers in blood, urine, and feces specimens has become an integral component of many epidemiologic studies; apart from these other well-known biological specimens have gained importance for clinical diagnostic purposes, disease evaluation, disease surveillance, or quality control of existing diagnostic testing procedures. To detail few of that kind of sample usage for CMD are mentioned below:

1.2.1. Lesion Swabs

• Vesicular lesions: Two swabs of vesicular fluid from an unopened vesicle, one for culture and one for real-time polymerase chain reaction (PCR).

• Eschars: Two saline-moistened swab samples, rotated underneath the eschar, one for culture and one for real-time PCR.

• Ulcers: Sample the base of the lesion with two saline-moistened swabs, one for culture and one for real-time PCR.

1.2.2. Saliva

• Clinically informative, biological fluid (biofluid) that is useful for novel approaches to prognosis, laboratory or clinical diagnosis, and monitoring and management of patients with both oral and systemic diseases.

• It is easily collected and stored and ideal for early detection of disease as it contains specific soluble biological markers (biomarkers).

• Saliva contains multiple biomarkers that make it useful for multiplexed assays that are being developed as point-of-care (POC) devices, rapid tests, or in more standardized formats for centralized clinical laboratory operations. Salivary diagnostics is a dynamic field that is being incorporated as part of disease diagnosis, clinical monitoring, and for making important clinical decisions for patient care (Tang et al., 2013; Lum and Le Marchand, 1998; Zhang and Ning, 2012).

1.2.3. Biopsy

• A full thickness biopsy of a papule or vesicle, including adjacent skin, for histopathology, special stains, and immunohistochemistry (IHC).

• For patients who are not on antibiotic therapy or who have been on therapy for <24  h, a second biopsy sample should be collected at the same time and submitted for culture and real-time PCR (Saunders and Lee, 2013; Schuller et al., 2010).

1.2.4. Serum

• An acute (≤7  days after symptom onset or as soon as possible after a known exposure event) serum sample to test for anthrax lethal factor toxin.

• Acute and convalescent (14–35  days after symptom onset) serum samples for serologic testing (Hsieh et al., 2006)

1.2.5. Plasma

• An acute plasma sample to test for anthrax lethal factor toxin (Alsaif et al., 2012)

• Plasma is the preferred specimen for anthrax lethal factor toxin testing (Zander et al., 2014)

1.2.6. Blood

• If there are signs of systemic anthrax infection (i.e., febrile or hypothermia, tachycardia, tachypnea, hypotensive), collect blood specimen before starting antimicrobial therapy for culture and real-time PCR.

1.2.7. Cerebrospinal Fluid

• To be submitted for patients with severe headache, meningeal signs, altered mental status, seizures, or focal signs for culture and real-time PCR.

1.2.8. Autopsy Tissues

• To be collected in fatal cases for histopathology, special stains, and IHC.

1.3. Cell Phenotype

Genome is dynamic and exquisitely sensitive, changing expression patterns in response to age, environmental stimuli, and pharmacological and physiological manipulations. Similarly, cellular phenotype, traditionally viewed as a stable end-state, should be viewed as versatile and changeable. The phenotype of a cell is better defined as a homeostatic phenotype implying plasticity resulting from a dynamically changing yet characteristic pattern of gene/protein expression. A stable change in phenotype is the result of the movement of a cell between different multidimensional identity spaces.

New platforms are designed for fast and high-throughput analysis of cellular phenotypes based on microscopy images. It is especially useful for large-scale profiling of cellular responses to pharmacological compounds, gene knockdowns, and/or toxic substances.

1.3.1. Cell-Based Diagnostic Methods

1.3.1.1. Fluorescence/Microscopic Assays

1.3.1.1.1. Single-Cell Phenotype Quantification

Automatically identifies individual cells from fluorescence microscopy images; and quantifies cellular morphology, protein subcellular localization, and other features.

1.3.1.1.2. Plate-Based Data Organization

Design to handle high volume of image data acquired from 96/384-well plates. Analyses of different fluorescent-marker configurations can be performed on the same plate data.

1.3.1.1.3. Quick-Plate Analysis

Perform cell count and fluorescence intensity measurements over the whole plates and visualize well-to-well trend and variability.

1.3.1.1.4. Phenotypic Profiling

Transform raw image features into discriminative profiles that can be used to characterize changes in cellular phenotypes.

1.3.2. Xenodiagnosis

1. A method of diagnosing acute or early Trypanosoma cruzi infection (Chagas disease) in humans. Infection-free (laboratory-reared) triatomine bugs are fed on the patient’s tissue, and the trypanosome is identified by microscopic examination of the bug’s intestinal contents after an incubation period.

2. A similar method of biologic diagnosis based on experimental exposure of a parasite-free normal host capable of allowing the organism in question to multiply, enabling it to be more easily and reliably detected.

1.3.3. Flow Cytometry

Flow cytometry is often used in conjunction with microscopy to characterize the diagnostics of the disease in more detail. For instance, flow cytometry can be used to identify cells expressing specific markers of disease that provide clues as to disease severity, patient prognosis, or optimal treatment. Host cells or infectious organisms are directly or indirectly (using dyes or markers) counted to assess the presence of disease. Flow cytometry is considered as a very quantitative and sensitive application. Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity, and localization of detected signals (i.e., surface, cytoplasm, nuclear). Most of the flow assays are initiated by preparing single cell or particle suspensions as necessary for flow cytometric analysis. Various immunoflurescent dyes or antibodies can be attached to the antigen or protein of interest. The suspension of cells or particles is aspirated into a flow cell where, surrounded by a narrow fluid stream, they pass one at a time through a focused laser beam. The light is either scattered or absorbed when it strikes a cell. Absorbed light of the appropriate wavelength may be reemitted as fluorescence if the cell contains a naturally fluorescent substance or one or more fluorochrome-labeled antibodies are attached to surface or internal cell structures. Light scatter is dependent on the internal structure of the cell and its size and shape. Fluorescent substances absorb light of an appropriate wavelength and reemit light of a different wavelength. Fluorescein isothiocyanate (FITC), Texas red, and phycoerythrin are the most common fluorescent dyes used in the biomedical sciences. Light and/or fluorescence scatter signals are detected by a series of photodiodes and amplified. Optical filters are essential to block unwanted light and permit light of the desired wavelength to reach the photodetector. The resulting electrical pulses are digitized, and the data is stored, analyzed, and displayed through a computer system. Modern flow cytometry allows single- or multiple-microbe detection (bacteria, fungi, parasites, and viruses) in an easy, reliable, and fast way on the basis of their peculiar cytometric parameters or by means of certain flourochromes that can be used either independently or bound to specific antibodies or oligonucleotides. In addition, flow cytometry has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Apart from these to provide few more flow applications with some examples are, Diagnosis of Hematologic Malignancies, Detection of Minimal Residual Disease, Lymphocyte Subset Enumeration, Analysis of DNA Ploidy and Cell Cycle, Efficacy of Cancer Chemotherapy, Reticulocyte Enumeration, Platelet Function Analysis, Cell function Analysis, Applications of Transfusion Medicine, and Applications in Organ Transplantation. However, no other laboratory instrument provides multiparametric analysis at the single cell level, and the flow cytometer or application variants of the flow cytometer will become more valuable as medical diagnosis and therapy changes. Different model-based cell line studies include tumor-immune-stromal, immune-stromal, tumor-immune-vascular, or immune-vascular microenvironments and disease relevant signaling pathways that manifest during tumorigenesis. By measuring and analyzing biomarker activities and/or comparing the resulting activity profiles of a test agent to the profiles of selected reference compounds or drugs, one can gain insight into the mechanism of action, efficacy, and safety-related effects (Table 1.1).

Table 1.1

Represents the biomarker analysis in different cell phenotypic profiling

Courtesy: Bodenmiller, B., 2016. Multiplexed epitope-based tissue imaging for discovery and healthcare applications. Cell Syst. 2 (4), 225–238.

1.4. Cell-Based Screening Assays

This screening-based technology covers a broad range of applications:

• Antimicrobial and antiviral compounds screening

• Toxic and nontoxic substances evaluation

• Detection of infectious viral contaminations

• Pathogen-associated molecular patterns (PAMPs) detection studies

• Pathogen-recognition-receptor agonists and antagonists screening

Compound screening process—Individual wells of a microplate are initially coated with a layer of living human cells, which are then incubated with the compounds to be tested as well as the respective pathogen. Normally, the pathogen immediately unfolds its pathogenic potential and kills the susceptible human cells. If, however, one of the compounds tested inhibits growth (proliferation) of the pathogen or blocks its virulence mechanisms, the human cells remain vital. Vital human cells can easily be detected photometrically using live dyes. Microscopic control of the individual wells of a microplate allows an additional evaluation of the physiological condition of the human cells. This results in another read-out parameter, which ensures that only well-tolerated and biocompatible compounds are selected for further testing. Generally applicable in antimicrobial drug discovery, it can also be applied to bacteria and fungi. This method is sensitive, robust, time- and cost-efficient, and especially effective in optimizing screening hits to lead structures (hit-to-lead optimization) and the development of candidates in the preclinical phase of development.

Pyrogenic residues detection process—This cell-based test system allows PAMPs to be identified and differentiated via their natural pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), NOD-like receptors, or dectins coupled to a reporter gene assay. PRRs are receptors of the human immune system that recognize components of viruses, bacteria, or fungi and normally initiate cytokine response. For this assay, the appropriate receptor complex (e.g., TLR2/6) is stably transfected and expressed in NIH3T3 fibroblasts. This cell line expresses no other PRR receptors and contains a reporter gene, which is induced by PRR activation. The induction of TLR2/6 for example by a specific ligand, Pam2CysSK4, leads to activation of the transcription factor NF-κB. This, in turn, induces the expression of the reporter gene, e.g., a secreted alkaline phosphatase. Pyrogens present in the analyte thus can be detected via expression of the reporter gene. Depending on the assay conditions, either formation of an insoluble deep blue precipitate that is easily detected visually or a high-throughput screening assay with photometric analysis can be performed. Cell-based test system allows fast and easy qualitative and quantitative detection of pyrogens without standard laboratory equipment. This assay enables screening for TLR antagonists that are increasingly used in dermatology in order to suppress immune reactions.

1.4.1. Plaque Assay

Cytopathogenic viruses can be quantified by the number of plaques or pocks they cause on susceptible cell monolayers. Using this assay, it will facilitate to screen drug compounds for plaque inhibition.

1.4.2. Tissue Culture Infectious Dose50 Assay

Viruses that have cytopathic effect can be quantitated using the tissue culture infectious dose50 Assay. Endpoint techniques are used for viruses that do not grow in culture, when Lethal Dose50 or Infectious Dose50 values must be calculated. They are also used in the case of viruses that are not cytopathic or do not produce plaques. This assay helps to determine the titer of cytopathogenic viruses in various samples.

1.5. Cytogenetics-Based Assays

1.5.1. Chromosome Analysis

Cytogenetics can be performed for the chromosome analysis on amniotic fluid, chorionic villus, and percutaneous umbilical blood sampling (PUBS) specimens.

Methodology—For amniotic fluid specimens, the in situ method of cell culture and clonal analysis is performed. G-banded metaphase spreads from 12 to 15 clones from a minimum of two independent cultures are examined. For chorionic villus sampling and PUBS samples, G-banded chromosomes from 20 metaphase cells are examined. Five cells are completely analyzed per sample and at least three karyograms are prepared. As per the need and conditions of experiment additional chromosome analysis and special banding techniques are employed.

Testing indications:

• Advanced maternal age

• Confirmation of an abnormal screening result

• Fetal abnormalities on ultrasound that are suggestive of a chromosomal defect

• Previous offspring with a chromosomal abnormality

• Family history of chromosomal abnormality

Analysis of abnormalities

Chromosome analysis detects:

• Aneuploidy (monosomy, trisomy)

• Deletions and duplications (>5  Mb)

• Translocations

• Inversions

• Chromosome analysis does not detect: Deletions or duplications that are too small to be seen by microscope (<5  Mb)

• Fluorescence in situ Hybridization (FISH)

Methodology

FISH utilizes DNA probes that bind to a specific region of a chromosome. These probes produce bright-colored signals that are examined on a fluorescence microscope. For metaphase FISH, 10 to 50 cells are scored (depending on the probe used). Rapid aneuploid screening utilizes fluorescent DNA probes, which bind to specific regions of chromosomes 13, 18, 21, X, and Y in interphase (nondividing) cells. These probes produce bright-colored signals that are examined under a fluorescence microscope. These probes are applied to amniotic fluid cells (no cell culture is necessary). Fifty interphase cells for each probe (13, 18, 21, X, and Y) are scored.

AneuVysion FISH (Titenko-Holland et al., 1994) can be performed on prenatal samples for rapid detection of the most common chromosome gains and losses seen prenatally (Table 1.2).

Testing indications:

• Late gestation pregnancies that require a rapid result

• Rapid confirmation of an abnormal prenatal screening result

• Detection of fetal abnormality on ultrasound suggests aneuploidy of chromosomes X, Y, 13 18, or 21

• Newborn with multiple anomalies suggests aneuploidy of chromosomes X, Y, 13, 18, or 21

Micro Deletion/Microduplication Syndrome Probes (Table 1.3)

Neoplastic FISH Panels (Table 1.4)

Table 1.2

Products of conception aneuploidy fluorescent in situ hybridization panel detects the most common chromosome gains and losses seen prenatally

Courtesy: Ramasamy, R., Scovell, J.M., Kovac, J.R., Cook, P.J., et al., 2015. Fluorescence in situ hybridization detects increased sperm aneuploidy in men with recurrent pregnancy loss. Fertil. Steril. 103 (4), 906–909.

Table 1.3

Represents the microdeletion/microduplication syndromes target profiling

Courtesy: Deak, K.L., Horn, S.R., Rehder, C.W., 2011. The evolving picture of microdeletion/microduplication syndromes in the age of microarray analysis: variable expressivity and genomic complexity. Clin. Lab. Med. 31 (4), 543–564.

Table 1.4

Represents the neoplastic fish target profiling

Courtesy: Shekhani, M.T., Barber, J.R., Bezerra, S.M., Heaphy, C.M., et al., 2016. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors. Hum. Pathol. 54, 106–112.

1.6. Cellular Pathways—Diagnostic Target Assays

This is a collection of cell-based pathway indicator assays designed to