327 Questions
What is the primary function of the CRISPR-associated protein (Cas) enzyme?
To cleave the invading DNA at specific locations
What is the main advantage of CRISPR/Cas9 over traditional restriction enzymes?
CRISPR/Cas9 provides the specificity of restriction enzymes with the versatility of guiding cuts to any sequence site
What is the primary purpose of the Southern blot?
To analyze specific DNA regions in a complex genome by RFLP
What is the primary goal of genomics?
To analyze hundreds to thousands of targets or whole genomes
What is the purpose of the spacer sequences in the CRISPR system?
To serve as adaptive immunity with memory of the invading DNA
What is the primary application of CRISPR/Cas9 in the laboratory?
To alter DNA at user-defined locations
What is the main advantage of CRISPR RNA over traditional restriction enzymes?
CRISPR RNA can lead transcription activators, repressors, gene promoters, or reporter molecules to target sequences
What is the purpose of a nucleating agent in liquid acrylamide solutions?
To facilitate the solidification of acrylamide
What is the primary function of a density agent in a loading solution?
To facilitate the loading of the sample into the wells
What is the purpose of a fluorescent stain in agarose gel electrophoresis?
To visualize the nucleic acid fragments
What is the primary advantage of capillary electrophoresis over agarose gel electrophoresis?
Increased sensitivity
What is the primary purpose of comparative genomic hybridization arrays?
To detect amplifications or deletions in DNA
What type of array is used to measure gene expression levels?
RNA expression arrays
What is the function of a platinum wire in agarose gel electrophoresis?
To establish a current through the gel
What is the term for the process of detecting chromosome microdeletions using microarrays?
Virtual karyotyping
What is the purpose of a molecular weight standard in agarose gel electrophoresis?
To estimate the size of the DNA fragments
What is the primary difference between agarose gel electrophoresis and capillary electrophoresis?
The presence of a gel matrix
What is the purpose of labeling the sample material in microarray analysis?
To allow for hybridization of the sample to the probes
What is the purpose of a laser in capillary electrophoresis?
To excite the fluorescent labels
What is the term for the type of array that can be used to determine DNA nucleotide sequence?
High-density oligonucleotide arrays
What is the purpose of using a reference material in microarray analysis?
To compare the levels of gene expression between the test and reference samples
What is the advantage of using multiple capillaries in capillary electrophoresis?
Ability to analyze multiple samples simultaneously
What is the purpose of a visual dye in a loading solution?
To visualize the sample during electrophoresis
What is the term for the type of array that uses beads as the solid support?
Bead array
What is the purpose of using fluorescent labels in bead array assays?
To detect the presence of the target protein or nucleic acid
What is the advantage of bead array assays over other types of arrays?
They can detect multiple targets simultaneously
What is a clinical application of bead array assays?
HLA typing
What is the primary function of restriction enzymes in DNA analysis?
To separate phosphodiester bonds between nucleotides
What is the purpose of molecular weight standards in capillary gel electrophoresis?
To enable instrument to automatically assess fragment size
What is the primary mechanism of DNA separation in capillary gel electrophoresis?
Electrokinetic process
What is the function of CRISPR-Cas9 system in DNA analysis?
To recognize and bind to specific nucleotide sequences
What is the purpose of formamide in preparing DNA samples for capillary gel electrophoresis?
To denature DNA into single strands
What is the result of DNA digestion with restriction enzymes?
Production of DNA fragments of varying sizes
What is the characteristic of DNA fragments produced by restriction enzymes?
Fragments are specific to the DNA sequence
What is the purpose of nucleic acid tests?
To detect changes in DNA sequence
What is the name of the first restriction enzyme isolated from E. coli?
EcoR1
What is the primary application of restriction fragment length polymorphism (RFLP) analysis?
All of the above
What is the primary purpose of electrophoresis in the analysis of nucleic acids?
To observe the sizes or amounts of nucleic acid
Which type of gel is more suitable for separating very large nucleic acids of tens of thousands of base pairs?
Agarose gel at low concentrations
Why do smaller nucleic acid chains move faster through the gel matrix during electrophoresis?
Because they have a smaller mass-to-charge ratio
What is the purpose of using a standard molecular weight marker in gel electrophoresis?
To estimate the size in bases or base pairs of the test nucleic acids
Which of the following is a characteristic of polyacrylamide gels?
More precisely designed for high-resolution separation
During electrophoresis, which pole do negatively charged nucleic acids move towards?
The anode
What determines the proper type and concentration of gel to be used in electrophoresis?
The expected sizes of the nucleic acids to be separated
Why are agarose gels less expensive and less toxic to use than polyacrylamide gels?
Because they are natural polymers
What is the primary function of oligonucleotide primers in a PCR reaction?
To ensure the specificity of the PCR reaction
What is the typical temperature range for the annealing step in a standard PCR cycle?
50°C–70°C
What is the purpose of the initial 5- to 15-minute incubation at the denaturation temperature in some PCR protocols?
To activate specialized DNA polymerases
What is the typical length of PCR products?
50 to more than 1,000 bp
What is the purpose of the final 7- to 10-minute step at the extension temperature in some PCR protocols?
To ensure complete copying of the products
What is the effect of ramp speed on PCR amplification?
It increases the efficiency of the amplification
What is the purpose of the denaturation step in a standard PCR cycle?
To separate the double-stranded DNA into single strands
What is the instrument used to carry out the amplification program in PCR?
Thermal cycler
What is the typical number of cycles in a PCR amplification program?
20 to 50 cycles
At what temperature is the extension step typically carried out in a standard PCR cycle?
68°C–72°C
What is the primary purpose of the internal amplification control in qPCR and RT-qPCR?
To verify that negative results are true negatives and not due to amplification failure
What is the primary difference between qPCR and digital droplet PCR?
The method of quantification of the target DNA
What is the purpose of the limiting dilution of sample template molecules in digital droplet PCR?
To prepare individual droplets of reaction buffer in oil
What is the primary application of qPCR in the detection of microorganisms?
To detect microorganisms that are difficult or dangerous to culture in the laboratory
What is the function of the fluorescent dye in the capillary gel electrophoresis detection of PCR products?
To label the PCR primer
What is the advantage of multiplex qPCR methods?
To assess multiple targets simultaneously
What is the primary difference between the lag phase and the log phase in the PCR curve?
The rate of PCR product accumulation
What is the purpose of the standard curve in qPCR?
To convert the Ct value to the number of DNA copies in the starting sample
What is the primary advantage of digital droplet PCR over qPCR?
It provides absolute quantification
What is the function of the emulsion formed in digital droplet PCR?
To act as individual reaction chambers
What is the primary purpose of gel or capillary gel electrophoresis in a PCR procedure?
To visualize the products of the PCR reaction
What is the role of reverse transcriptase in RT-PCR?
To synthesize cDNA from RNA
What is the purpose of the 3′ end of the primer in SSP-PCR?
To anneal to the template on a potentially mutated or polymorphic base pair
What is the advantage of using probes in qPCR compared to SYBR green?
Probes provide higher specificity for the intended product
What is the purpose of the 5′ end of the primer in PCR?
To allow for the attachment of noncomplementary sequences containing restriction enzyme recognition sites
What is the difference between RT-PCR and qPCR?
RT-PCR is used for qualitative detection, while qPCR is used for quantitative detection
What is the purpose of FRET, TaqMan, molecular beacon, and scorpion probes in qPCR?
To hybridize to the template by sequence complementarity and generate fluorescent signals
What is the advantage of using qPCR compared to standard PCR?
qPCR allows for the quantification of starting material
What is the purpose of SYBR green in qPCR?
To detect the accumulation of PCR product in real time
What is the purpose of sequence-specific primer PCR (SSP-PCR)?
To detect mutations and polymorphisms
What is the primary function of RNase II in the process of probe amplification?
To degrade hybridized RNA, leaving single-stranded DNA
In the process of strand displacement amplification, what is the function of the outer primer?
To bind to the target DNA and extend, displacing the product formed by the simultaneous extension of the inner primer
What is the primary advantage of loop-mediated isothermal amplification (LAMP)?
Ability to amplify target DNA in a short amount of time
In molecular inversion probe (MIP) amplification, what happens to the probe ends when they bind to target sequences?
They are brought together and ligated to form circles
What is the primary function of branched DNA (bDNA) amplification?
To capture the target nucleic acid and bind to multiple reporter molecules
What is the main advantage of branched DNA (bDNA) amplification over methods using a single probe or primer?
Higher specificity due to the use of multiple probes
What is the primary function of DNA?
To store genetic information
What is the primary difference between probe amplification and signal amplification?
Probe amplification amplifies the target DNA sequence, while signal amplification amplifies the signal bound to the target
What is the purpose of the restriction enzyme in strand displacement amplification (SDA)?
To form a nick (cutting only one strand of the double helix) in the double-stranded DNA segment
Which of the following sequencing methods uses modified nucleotide bases called dideoxynucleotide triphosphates (ddNTPs)?
Chain termination sequencing
What is the primary application of molecular inversion probe (MIP) amplification?
All of the above
What is the result of the sequencing reaction in Sanger sequencing?
A collection of fragments of various sizes
What is the advantage of using multiple probes in branched DNA (bDNA) amplification?
Enhanced specificity due to the use of multiple probes
What is the purpose of using fluorescent labels in Sanger sequencing?
To distinguish between the different ddNTPs
What is the purpose of resolving the DNA ladder by gel or capillary gel electrophoresis?
To separate the DNA fragments based on their size
What is the term for the collection of fluorescent peaks obtained by capillary gel electrophoresis?
Electropherogram
What is the purpose of the primer pair in Sanger sequencing?
To outline the target DNA
What is the reason why synthesis will stop if a ddNTP is incorporated into the growing DNA chain?
Because the ddNTP lacks an OH group at the 3′ carbon of the deoxyribose sugar
What is the advantage of capillary electrophoresis over gel electrophoresis?
It is faster and more automated
What is the purpose of bDNA signal amplification assay?
To detect and quantify hepatitis B virus, HCV, and HIV-1
What is the primary purpose of including positive and negative controls in in situ hybridization (ISH) testing?
To ensure the accuracy of the results and verify tissue preparation
What is the main function of programmed cell death ligand (PD-L1) in the immune response?
To suppress the adaptive immune response by binding to programmed cell death protein 1 (PD-1) on activated lymphocytes and dendritic cells
What is the primary difference between fluorescence in situ hybridization (FISH) and in situ hybridization (ISH) methods?
FISH requires specialized microscopes equipped to detect fluorescent signals, whereas ISH does not
What is the purpose of using reference probes in fluorescence in situ hybridization (FISH) methods?
To identify the chromosomes of interest while assessing deletion or amplification
What is the term used to describe the sensitivity of in situ hybridization (ISH) methods to the buffer and temperature conditions of hybridization?
Stringency
What is the main advantage of using array methods in molecular diagnostics?
They can detect multiple or complex genetic abnormalities
What is the primary purpose of amplification methods in molecular diagnostics?
To copy nucleic acids for further analysis
Who is credited with the development of the in vitro PCR, which greatly facilitated and broadened the potential applications of gene amplification?
Kerry Mullis
What is the primary purpose of using immunohistochemistry in in situ hybridization (ISH) testing?
To detect the presence of clinically significant protein targets, such as those expressed by tumor cells
What is the term used to describe the process of detecting targets in place as they appear in tissues, cells, and subcellular structures?
In situ hybridization (ISH)
What is the primary mechanism by which TaqMan probes generate a signal?
The target DNA is copied, releasing the reporter dye from the quencher.
What is the key difference between molecular beacons and TaqMan probes?
Molecular beacons have a quencher and a reporter dye, but they are not separated in the presence of target sequences.
What is the purpose of the scorpion probe?
To generate a signal as the target DNA is copied, with the reporter covalently attached to the product.
What is the primary advantage of digital droplet PCR over qPCR?
It is more sensitive and can detect smaller changes in target number.
What is the primary mechanism of transcription-based amplification systems?
A complementary DNA copy is synthesized from the target RNA, and then transcription of the cDNA produces millions of copies of RNA products.
What is the primary advantage of targeting RNA in transcription-based amplification systems?
It is more sensitive than targeting DNA because each microorganism makes multiple copies of RNA.
What is the primary application of transcription-mediated amplification (TMA)?
Detection of RNA viruses, such as HCV and HIV.
What is the purpose of the cDNA:RNA hybrid in transcription-mediated amplification?
To form a template for the synthesis of more cDNA.
What is the advantage of TMA over PCR?
TMA is an isothermal process, which does not involve the repeated heating and cooling required for PCR.
What is the primary application of digital PCR in infectious disease?
Detection of rare mutations in infectious diseases.
What is the main purpose of Sanger sequencing in genetics and oncology?
To identify gene abnormalities in inherited diseases
Why is pyrosequencing less sensitive than other methods for detecting DNA sequence changes?
Because it is affected by the heterozygous form of the alteration
What is the purpose of sequencing the complementary strand of the DNA in Sanger sequencing?
To confirm the alteration detected in the original sequence
What is the advantage of pyrosequencing over Sanger sequencing?
It is less labor-intensive and more convenient for short sequence analyses
What is the purpose of the sequencing primer in pyrosequencing?
To initiate the DNA synthesis reaction
What is the result of the pyrosequencing reaction?
A pyrogram of luminescent peaks
Why is pyrosequencing used in genetics and oncology?
To sequence targeted areas of known gene locations
What is the limitation of pyrosequencing compared to Sanger sequencing?
It produces shorter sequence reads
What is the purpose of adenosine 5′ phosphosulfate (APS) in pyrosequencing?
To convert pyrophosphate to ATP
What is the advantage of using some instruments developed for genomic or next-generation sequencing (NGS) in pyrosequencing?
They utilize the pyrosequencing chemistry
What was the primary goal of developing Next-Generation Sequencing (NGS) technologies?
To sequence the human genome for a minimal cost of less than $1,000
What is a characteristic of NGS procedures?
Starting with short DNA templates, usually less than 500 bp
Which of the following NGS technologies has been most frequently applied to clinical applications?
Pyrosequencing, reversible dye sequencing, and ion conductance sequencing
What was the primary challenge that was not initially included in the sequencing cost of NGS technologies?
The cost of interpreting the data
What is the primary difference between NGS and traditional Sanger sequencing?
The number of genomes that can be sequenced simultaneously
What is the term for the goal of sequencing the human genome for a minimal cost of less than $1,000?
The $1,000 genome
What is the primary benefit of using large databases of somatic variant data in cancer diagnosis and treatment?
To refine interpretation of newly collected data
What is the primary advantage of NGS technologies over traditional Sanger sequencing?
Lower cost of sequencing
What is the name of the project that has sequenced thousands of genomes using NGS technologies?
The 1,000 Genome Project
What is the primary advantage of using NGS over Sanger sequencing?
NGS provides higher coverage of regions of interest
What is the purpose of using quality, variant type, and allele frequency parameters in filtering?
To identify the most biologically significant variants
Why is adequate coverage of regions of interest required in NGS?
To provide sufficient data for confident base calling
What is the primary purpose of annotating variants using historical sequence database information?
To determine the medical significance of variants
What is the primary benefit of using a standard nomenclature system for sequence information?
To facilitate clear communication and organized storage of sequence data
What is the primary purpose of using targeted gene panels in NGS?
To produce a finished report with variants identified automatically
Why are errors more common in NGS compared to Sanger sequencing?
Library preparation is more error-prone in NGS
What is the primary purpose of using FASTQ files in NGS?
To include a quality symbol for each base
What is the primary benefit of using bioinformatics in molecular analyses?
To facilitate the interpretation of large amounts of data
What is the primary purpose of comparing the sequence of a DNA sample to a reference sequence in NGS?
To identify variants or polymorphisms in the sample
What is the minimum coverage required to call rare variants in NGS?
500X
What is the primary application of targeted NGS in the clinical laboratory?
HLA typing, oncology, and genetics
What is the purpose of the Cancer Genome Atlas (TCGA) program?
To understand the genetic basis of cancer and improve diagnosis and treatment
What is the term for the number of mutations per megabase of sequenced DNA in a tumor?
Tumor mutational burden
What is the primary advantage of NGS over other sequencing technologies?
Ability to investigate all known genetic loci
What is the purpose of sequencing gene panels in NGS?
To identify genetic variants in a specific set of genes
What is the term for the percentage of sequences carrying a variant in NGS?
Allele frequency
What is the primary advantage of NGS in HLA typing?
Ability to resolve ambiguities
What is the primary purpose of using PCR primers or probes in NGS?
To select and copy targeted regions for sequencing
What is the primary factor that limits the degree of coverage used in NGS?
Cost
What is the function of the bar code or index in NGS?
To identify each sample and gene region in the library
What is the purpose of emulsion PCR in NGS?
To generate a library of PCR products attached to beads
What is the mechanism of detection in ion conductance sequencing?
Release of hydrogen ions and detection of pH change
What is the purpose of bridge PCR in reversible dye terminator technology?
To form clusters of templates across the flow cell
What is the advantage of ion conductance sequencing over other methods?
It is faster and does not require optical sensing
What is the purpose of adapters in NGS?
To carry PCR primer-binding sites and allow unbiased amplification
What is the purpose of the sequencing reagents in pyrosequencing?
To release PPi and generate light signals
What is the advantage of using multiple samples in a single run of NGS?
It decreases the cost and time required for sequencing
What is the purpose of the picoplate in pyrosequencing?
To carry out independent sequencing reactions in hundreds of thousands of wells
What is the primary function of flow cytometry in the clinical laboratory?
To monitor HIV infection status and enumerate hematopoietic stem cells
What is the primary purpose of using fluorescent-labeled antibodies or probes in flow cytometry?
To identify specific surface or cytoplasmic proteins
What is a characteristic of fluorochromes used in flow cytometry?
They absorb light across a spectrum of wavelengths and emit light of lower energy across a spectrum of longer wavelengths
What is the primary application of flow cytometry in the diagnosis of diseases?
In the diagnosis of leukemia and lymphoma
What is the primary advantage of using multiple fluorochromes in flow cytometry?
It allows for the analysis of multiple cell properties simultaneously
What is the primary function of laser light in flow cytometry?
To excite fluorochromes and detect cell properties
What is the primary application of flow cytometry in transplantation?
To detect human leukocyte antigen (HLA) antibodies
What is the primary advantage of using flow cytometry in cell analysis?
It allows for the analysis of a large number of cells simultaneously
What is the primary purpose of using optical filters in flow cytometry?
To maximize collection of light from specific fluorochromes
What is the effect of using Ficoll-Hypaque density gradient centrifugation on some cell populations?
Selective loss of some cell populations
What is the purpose of using erythrocyte lysis techniques in sample preparation for flow cytometry?
To destroy erythrocytes while leaving WBCs intact
What is the typical range of digital signals measured in flow cytometry?
1 to 256 channels
What is the purpose of using a marker in flow cytometry data analysis?
To differentiate cells with low levels of fluorescence from cells with high levels of fluorescence
What is the primary purpose of using fiber-optic cables in flow cytometry?
To direct light to the photomultiplier tubes
What is the effect of using heparin as an anticoagulant in sample preparation for flow cytometry?
Improved stability of samples for up to 48 hours
What is the primary purpose of using EDTA as an anticoagulant in sample preparation for flow cytometry?
To collect whole blood samples for analysis within 30 hours
What is the typical number of events collected for each sample in flow cytometry data acquisition?
10,000 to 20,000 events
What is the primary purpose of using a single-parameter histogram in flow cytometry data analysis?
To plot a chosen parameter versus the number of events
What is the primary advantage of flow cytometry?
It enables the accurate detection of cells that are present in very small numbers.
What is the purpose of the fluidics system in a flow cytometer?
To ensure that cells pass through the laser light one cell at a time.
What determines which fluorochromes can be used in an assay?
The wavelength of the laser light.
What is the primary defect in patients with paroxysmal nocturnal hemoglobinuria (PNH)?
Lack of GPI anchor proteins on the cell surface
Which of the following tests can detect minimal residual disease (MRD) in patients with leukemia or lymphoma?
Flow cytometry
What is the difference between forward scatter and side scatter?
Forward scatter measures size, while side scatter measures granularity.
What is the purpose of using fluorescent-labeled antibodies in flow cytometry?
To detect the presence of specific antigens on the surface of cells.
What is the primary application of flow cytometry in transplant immunology?
HLA typing and cross-matching
What is the advantage of using multiple lasers in a flow cytometer?
It enables the analysis of up to five fluorochromes at a time.
What is the advantage of using monoclonal antibodies over polyclonal antibodies in immunophenotyping?
Monoclonal antibodies are more specific
What is the primary function of cytometric bead arrays?
Identifying multiple analytes simultaneously
What is the purpose of the optics and photodetectors in a flow cytometer?
To detect the light scatter and fluorescence signals generated by the cells.
What is the term for the parameters that are intrinsic to the cell?
Intrinsic parameters.
Which of the following is an application of flow cytometry in molecular diagnostics?
Detecting viral nucleic acids
What is the advantage of using multicolor analysis in flow cytometry?
It enables the analysis of multiple extrinsic parameters simultaneously.
What is the primary advantage of flow cytometry over traditional serological methods?
Faster and more accurate results
What is the purpose of analyzing the data generated by the flow cytometer in conjunction with forward scatter and side scatter?
To enable more accurate subtyping of cells.
What is the purpose of fluorescent labels in bead array assays?
To detect analytes
Which of the following is a clinical application of flow cytometry?
All of the above
What is the primary purpose of a gate in a dual-parameter dot plot?
To filter out debris and isolate subpopulations of cells of interest
What is the primary advantage of using flow cytometry in DNA analysis?
All of the above
What is the advantage of using a single-platform analysis over a dual-platform analysis?
It eliminates the potential for added error associated with the use of two distinct methods
What is the primary purpose of immunophenotyping in the clinical laboratory?
To characterize and enumerate normal lymphocytes
What is the primary difference between quadrants 1 and 2 in a dual-parameter dot plot?
Quadrant 1 consists of cells that are positive for fluorescence on the y-axis and negative for fluorescence on the x-axis, while quadrant 2 consists of cells that are positive for fluorescence on both the x- and y-axes
What is the primary purpose of the computer and specialized software in a dual-parameter dot plot analysis?
To calculate the percentage of cells in each quadrant
What is the primary advantage of using bead-based methods over volumetric methods in single-platform analysis?
Bead-based methods provide a more accurate absolute count of a particular cell type
What is the primary purpose of detailed phenotypic analysis in the clinical laboratory?
To determine the degree of differentiation and activation of a specific cell population
What is the primary difference between nonmalignant immunophenotyping and malignant immunophenotyping?
Nonmalignant immunophenotyping is used to characterize and enumerate normal lymphocytes, while malignant immunophenotyping is used to diagnose malignant disorders
What is the primary purpose of using a known quantity of fluorescent beads in a bead-based method?
To provide a simple mathematic calculation to directly determine the absolute WBC numbers
What is the primary advantage of using a single-platform analysis over a traditional method?
It eliminates the potential for added error associated with the use of two distinct methods
What is the primary purpose of immunophenotyping by flow cytometry in leukemia and lymphoma management?
To identify the lineage of hematopoietic malignancies
Which of the following markers is typically paired with CD20 in the analysis of chronic lymphocytic leukemia (CLL)?
CD5
What is the significance of CD45 expression levels in flow cytometry analysis?
It is used to differentiate various populations of WBCs
What is the primary application of flow cytometry in the evaluation of HIV disease?
To classify stages of HIV disease and determine treatment options
What is the characteristic of neutrophils from patients with chronic granulomatous disease (CGD)?
They are unable to oxidize the dye dihydrorhodamine 123
What is the purpose of examining the FSC and SSC plot in flow cytometry analysis?
To ensure that a population is not being missed
What is the significance of CD4+ T-cell numbers in HIV-infected patients?
They are used to classify stages of HIV disease and determine treatment options
What is the primary purpose of immunophenotyping in the evaluation of immunodeficiency?
To compare patient values with reference ranges
What is the significance of CD8+ T-cell levels in HIV-infected patients?
They are expanded during the early course of HIV disease
What is the characteristic of blasts in terms of CD45 expression?
They have low levels of CD45 expression
What was the primary motivation for the development of immunoassay analyzers?
To create an automated system capable of reducing or eliminating manual tasks
What is a benefit of immunoassay automation in addition to reducing error and increasing accuracy?
The ability to provide more services with less staff
What is a challenge of selecting an immunoassay analyzer for a laboratory?
The difficulty of determining which analyzer is best suited for a laboratory's needs
What is a benefit of immunoassay automation in terms of reagent management?
The longer shelf life of reagents
What is a common type of analyte that can be measured using immunoassay automation?
Low-level peptides such as peptide hormones
How many different types of automated immunoassay analyzers are available?
More than 60
What is a consequence of eliminating manual steps in immunoassay testing?
The decrease in the likelihood of error
What is a potential benefit of immunoassay automation in terms of sample management?
Sample delivery with bar codes for better sample identification
What is a primary limitation of batch analyzers?
They can only perform one type of analysis at a time
What is the purpose of clot detectors in stainless-steel probes?
To reject samples with clots
What is a common issue associated with reusable pipette probes?
Carryover or contamination of samples
What is the primary purpose of reagent storage compartments in some analyzers?
To store reagents at a controlled temperature
What is the purpose of magnetic stirring in analyzers?
To mix samples with reagents
What is the primary purpose of timed incubation in analyzers?
To incubate samples at a controlled temperature
What is the primary advantage of using batch analyzers in clinical laboratories?
Cost-effectiveness when performing a large scale of a single type of testing
What is the primary advantage of random-access analyzers over batch analyzers?
They can perform multiple analyses on a single sample
What is the purpose of validating new instrumentation or methodology in clinical laboratories?
To meet the Clinical Laboratory Improvement Amendments (CLIA) regulations
What is the primary purpose of fluorescent detectors in analyzers?
To measure the concentration of analytes in a sample
What is the primary purpose of automation in analyzers?
To reduce the need for manual sampling
Which of the following is a required verification for a new method in clinical laboratories?
All of the above
What is the primary purpose of liquid-level sensors in analyzers?
To detect the lack of sample in a tube
What is the definition of analytic specificity in clinical laboratory testing?
The ability to generate a negative result when the analyte is not present
What is the purpose of parallel testing with an alternative method or technology in clinical laboratory testing?
To assess the accuracy of an assay
What is the advantage of using random-access analyzers in clinical laboratories?
Flexibility in rapid processing of stat samples
What is the purpose of the Centers for Medicare and Medicaid Services (CMS) in relation to clinical laboratory testing?
To provide an overview of the Clinical Laboratory Improvement Amendments (CLIA) regulations
What is the definition of accuracy in clinical laboratory testing?
The ability to measure what the test claims to measure
What is the purpose of calculating the standard deviation and coefficient of variation in clinical laboratory testing?
To evaluate the precision of an assay
What is the purpose of including at least one normal and one abnormal control in the analysis of precision in clinical laboratory testing?
To ensure the precision of an assay is determined under different conditions
Which of the following is NOT a nitrogen base found in nucleic acids?
Methionine (M)
What is the primary function of DNA polymerase enzyme?
To catalyze the synthesis of DNA
What is the term for a change in the nucleotide sequence of DNA?
Mutation
What is the primary difference between DNA and RNA?
DNA is mostly double-stranded, while RNA is often single-stranded
What is the purpose of a primer in DNA replication?
To provide the necessary 3' hydroxyl group for DNA replication
What is the process by which genetic information flows from DNA to mRNA?
Transcription
What is the result of a change in the nucleotide sequence of DNA, depending on its frequency?
A mutation or a polymorphism
What is the purpose of RNA polymerase enzyme in RNA synthesis?
To catalyze the synthesis of RNA
What is the main difference between capillary electrophoresis and agarose gel electrophoresis?
The type of gel used
What is the primary purpose of hybridization in microarray analysis?
To bind two complementary strands of nucleic acids
What is the main advantage of microarray analysis over other methods?
It can detect expression of thousands of genes simultaneously
What is the primary function of restriction enzymes in DNA analysis?
To isolate specific parts of a DNA sequence for analysis
What is the purpose of fluorescent labels in microarray analysis?
To aid in the detection of hybridized DNA
What is the main limitation of microarray analysis?
It is expensive and not efficient compared to auto sequencing
What is the primary application of microarray analysis?
Biomedical research, clinical diagnosis, and pharmaceuticals
What is the purpose of submitting microarray data to the Gene Expression Omnibus Server?
To make the data available to researchers worldwide
What is the primary mechanism of DNA separation in electrophoresis?
Charge of the DNA molecules
What is the purpose of normalization in microarray data analysis?
To adjust for differences in intensity
What is the primary function of the dichroic mirror in a fluorescence microscope?
To reflect light only at the excitation wavelength
What is the main reason for using a mercury arc lamp as the light source in fluorescence microscopy?
It provides a high-intensity, wide wavelength range of light
What is the term for the phenomenon where the peak of absorption and emission curves differ?
Stoke's shift
What is the purpose of adding an anti-fade mounting medium to the slide in fluorescence microscopy?
To reduce photobleaching of the fluorophore
What is the primary function of the barrier filter in a fluorescence microscope?
To filter out contaminating light from the arc lamp
What is the term for the weakening or loss of fluorescence due to prolonged excitation?
Photobleaching
What is the primary advantage of fluorescence microscopy over traditional light microscopy?
Ability to detect specific cell types or molecules
What is the term for the component that combines the exciter filter, dichroic mirror, and barrier filter in a fluorescence microscope?
Filter cube
What determines the excitation wavelength used in fluorescence microscopy?
The type of fluorophore used
What is the primary purpose of the objective lens in a fluorescence microscope?
To focus the excitation light onto the sample
What is the primary mechanism by which FISH exploits the ability of DNA strands to hybridize specifically?
Complementary sequences find each other and bind together
What is the main advantage of using RNA probes in FISH?
All of the above
What is the primary purpose of the dehydration step in FISH?
To facilitate hybridization of the probe and target DNA
What is the main application of FISH in karyotyping and phylogenetic analysis?
To determine the evolutionary relationships between organisms
What is the primary difference between FISH and FANA?
FISH uses fluorescent probes, while FANA uses radioactive probes
What is the primary purpose of the Stoke's shift in fluorescence microscopy?
To emit light at a different wavelength
What is the main advantage of using synthetic oligonucleotide probes in FISH?
All of the above
What is the primary purpose of the xenon or mercury light source in fluorescence microscopy?
To provide a powerful light source for excitation
What is the main advantage of using fluorescent labels in FISH?
They are more sensitive than radioactive labels
What is the primary application of fluorescence microscopy in FISH?
To detect specific DNA sequences
What is the primary purpose of using an ENA panel in autoimmune disease diagnosis?
To rule out other autoimmune disorders, such as Sjörgen syndrome, polymyositis, and scleroderma
What is the 'gold standard' method for detecting ANA?
Indirect fluorescent antibody (IFA)
What is the unit of measure reported in an immunoassay test for ANA?
U (arbitrary unit)
What is the pattern of cellular fluorescence associated with SLE and mixed connective tissue disease?
Homogeneous (diffuse)
What is the significance of a high titer in an IFA test for ANA?
The person is more likely to have an autoimmune disease
What is the most common condition associated with a positive ANA test?
SLE
What is the primary purpose of using additional laboratory tests, such as ESR and CRP, in autoimmune disease diagnosis?
To evaluate a person for inflammation
What is the significance of a positive ANA test result in a person without symptoms?
It may take time to determine the significance of the result
What is the purpose of reporting the pattern of cellular fluorescence in an IFA test for ANA?
To associate the ANA with a specific autoimmune disorder
What is the significance of a negative ANA test result in a person with symptoms of an autoimmune disease?
The test is likely to be a false negative
What percentage of people with Sjörgen syndrome have a positive ANA test result?
40-70%
What is the primary application of flow cytometry in hematology?
Immunophenotyping of whole blood and bone marrow
What is the purpose of the Kleihauer-Betke acid-elution test?
To detect fetal red cells in maternal blood
What is the advantage of using flow cytometry over traditional methods?
It provides rapid analysis of multiple characteristics of single cells
What percentage of people with scleroderma have a positive ANA test result?
60-90%
What is the primary advantage of flow cytometry in the clinical laboratory?
It is more user-friendly and has a wider range of clinical applications
What is the purpose of flow cytometry in immunophenotyping?
To analyze cell surface and cytoplasmic antigens
What is the primary application of flow cytometry in the detection of fetal red cells in maternal blood?
To detect the presence of fetal red cells
What is the advantage of using monoclonal antibodies in flow cytometry?
They are more specific than polyclonal antibodies
What is the primary purpose of using Rh-immune globulin in pregnant women?
To prevent alloimmunization
What is the primary advantage of using flow cytometry for the detection of fetal cells compared to the Kleihauer-Betke test?
More objective, reproducible, and sensitive results
What is the primary advantage of using fluorescent dyes that bind to residual RNA in reticulocyte counting?
Excellent discrimination between reticulocytes and mature RBCs
What is the primary importance of CD4 counts in HIV-infected individuals?
Assessing immune status
What is the primary advantage of using flow cytometry in immunophenotyping of leukemias and lymphomas?
Rapid and effective diagnosis
What is the primary application of flow cytometry in hematology?
All of the above
What is the primary advantage of using antibodies to hemoglobin F in fetal cell detection?
More objective and sensitive results
What is the primary importance of CD4 counts in HIV-infected individuals, compared to viral load testing?
CD4 counts directly assess immune status, while viral load testing measures viral load
What is the primary advantage of using flow cytometry in distinguishing myeloid and lymphoid lineages in acute leukemias?
More effective distinction between lineages
What is the primary application of flow cytometry in the diagnosis of leukemias and lymphomas?
All of the above
What is the primary advantage of using flow cytometry in hematology, compared to traditional methods?
More objective, reproducible, and sensitive results
What is the purpose of allowing the xenon or mercury light source to warm up for 15 minutes?
To allow the light source to reach constant illumination
What is the function of the filter cube in fluorescence microscopy?
To select the correct wavelength for the fluorescent dye
What is the purpose of using antibodies conjugated to fluorescent compounds in fluorescence microscopy?
To label specific proteins of interest
What is the advantage of using fluorescence recovery after photobleaching (FRAP) over standard fluorescence microscopy?
It enables the study of protein dynamics and kinetics
What is the primary application of fluorescent microscopy in the study of autoimmune disorders?
Imaging of proteins labeled with antibodies conjugated to fluorescent compounds
What is the purpose of the antinuclear antibody (ANA) test?
To diagnose autoimmune disorders
What is the difference between incident light and emitted light in fluorescence microscopy?
Incident light has a shorter wavelength, while emitted light has a longer wavelength
What is the purpose of using fluorescently labeled macromolecular assemblies in fluorescence speckle microscopy?
To study the movement and turnover kinetics of cytoskeletal proteins
What is the advantage of using fluorophores with different emission wavelengths in fluorescence microscopy?
It allows for the imaging of multiple structures or molecules simultaneously
What is the purpose of keeping the exposure time constant when comparing features with the same dye on different samples?
To ensure consistent imaging results
Study Notes
Analysis of DNA Mutations and Polymorphisms
- Analysis of DNA mutations and polymorphisms involves various laboratory procedures that use electrophoresis to observe the sizes or amounts of nucleic acid.
- Electrophoresis is the movement of particles under the force of an electric current, and it can be used to separate particles in gas, liquid, or solid phases.
Gel Electrophoresis
- Gel electrophoresis is a type of electrophoresis that uses a semisolid matrix or gel to sieve the nucleic acid polymers.
- There are two types of gels used for nucleic acid analysis: agarose and polyacrylamide.
- Agarose gels are natural polymers of agarobiose, a disaccharide found in plants, and are useful for standard laboratory separations of nucleic acids of 50 bp or more.
- Polyacrylamide gels are synthetic polymers of acrylamide and bis-acrylamide, and are more precisely designed for high-resolution separation, distinguishing differences in nucleic acids as small as one nucleotide.
Capillary Electrophoresis
- Capillary electrophoresis is a more sensitive, semiautomated type of electrophoresis that separates particles in a gas, liquid, or gel.
- It uses a gel or polymer inserted into the capillary to sieve the nucleic acids, and can detect fluorescent signals at more than one wavelength.
- The instrument computes and displays the lengths of the nucleic acid fragments in base pairs.
Molecular Analysis
- Nucleic acid tests are designed to detect changes in the DNA sequence (mutations and polymorphisms) or to measure differences in amounts of RNA synthesized.
- There are four main approaches to nucleic acid analysis: strand cleavage methods, hybridization methods, amplification methods, and sequencing.
Strand Cleavage Methods
- Restriction enzyme mapping is a method that uses restriction enzymes to cleave DNA at specific sites, and the resulting fragments are separated by electrophoresis.
- Restriction enzymes are endonucleases that recognize and bind to specific nucleotide sequences in the DNA, and are named for the organisms from which they are isolated.
- CRISPR-Cas9 is a type of restriction system that uses a common enzyme guided by RNA to specific sites, and has been used in the laboratory to alter DNA at user-defined locations.
Hybridization Methods
- Hybridization involves the binding of two complementary strands of nucleic acids, and is used to identify the region of interest in a complex genome.
- Southern blot is a method that uses restriction enzymes to cleave the DNA into smaller fragments, which are then separated by electrophoresis and identified using labeled probes.
- Northern blot is a variation of the Southern blot that is used to analyze RNA structure and expression.
Array Methods
- Array methods involve the simultaneous analysis of many genes or proteins to assess the true biological state of a cell or an organism.
- There are three basic types of arrays: comparative genomic arrays, RNA expression arrays, and high-density oligonucleotide or SNP arrays.
- Microarrays use highly specific unlabeled probes attached directly to a solid support, and are used for a variety of applications, including detection of chromosome microdeletions and gene-expression profiling.
In Situ Hybridization
- In situ hybridization (ISH) refers to the detection of targets in place as they appear in tissues, cells, and subcellular structures.
- Labeled probes are used to bind or hybridize to the targets, and are frequently used in pathology studies of tissue and cell suspensions.
- Immunohistochemistry is a type of ISH that uses labeled antibodies to detect the presence of clinically significant protein targets.### Fluorescence in Situ Hybridization (FISH)
- FISH is a technique used to detect and localize specific DNA sequences in chromosomes or cells
- Probes range in size from a few thousand to hundreds of thousands of bases long and are covalently attached to a fluorescent dye
- The technique can be performed on non-dividing (interphase) cells or directly on metaphase chromosomes from dividing cells
- The DNA from the sample is denatured into single strands, and the probes are applied to prepared slides of the cells or chromosomes, where they hybridize to their complementary sequences
- The resulting signals indicate if the targeted gene or region is abnormal
Amplification Methods
- Amplification methods involve the copying of nucleic acids
- The most frequently used methods involve some aspect of amplification, such as PCR (Polymerase Chain Reaction)
- Other target-amplification methods include reverse transcriptase PCR (RT-PCR), transcription-mediated amplification (TMA), and strand displacement amplification (SDA)
Polymerase Chain Reaction (PCR)
- PCR is an in vitro DNA replication procedure
- A PCR reaction includes all the necessary components required for DNA replication, such as the sample containing the DNA template, oligonucleotide primers, deoxyribonucleotides, DNA polymerase, and buffer
- The oligonucleotide primers are key components of the PCR reaction, as they provide specificity
- The PCR reaction mix is subjected to an amplification program consisting of a designated number of cycles, with each cycle consisting of three steps: denaturation, annealing, and extension
- The resulting PCR products are visualized by gel or capillary gel electrophoresis
PCR Variations
- Reverse transcriptase PCR (RT-PCR) starts with an RNA template and involves the synthesis of complementary DNA (cDNA) from the RNA
- Sequence-specific primer PCR (SSP-PCR) involves the use of primers that are designed to end on a potentially mutated or polymorphic base pair
- Quantitative PCR (qPCR) involves the measurement of the accumulation of PCR product in real-time during amplification
- Digital droplet PCR is a method that provides absolute quantification of the number of molecules in the sample
Transcription-Based Amplification
- Transcription-based amplification methods, such as transcription-mediated amplification (TMA), involve the synthesis of millions of copies of RNA products from a target RNA
- These methods are isothermal, meaning they do not require the repeated heating and cooling required for PCR
- TMA is suitable for the detection of RNA viruses, such as HIV and HCV, and for the detection of organisms with DNA genomes, such as Mycobacterium tuberculosis
Probe Amplification
- In probe amplification, the number of target nucleic acid sequences in a sample is not changed, but rather primers are extended or ligated into many copies of detectable probes
- Examples of probe amplification methods include strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and molecular inversion probe amplification (MIP)
- These methods are highly sensitive and specific, and are used for the detection of a variety of targets, including viruses, bacteria, and genetic mutations.### Signal Amplification
- Signal amplification involves binding large amounts of signal to target sequences in a sample.
- Branched DNA (bDNA) is a commercially available signal amplification method that uses short, single-stranded DNA probes to capture target nucleic acid and bind to multiple reporter molecules.
- bDNA amplification enhances specificity by allowing multiple probes to hybridize to target sequences, enabling detection of multiple genotypes of the same virus.
DNA Sequencing
- DNA sequencing determines the order or sequence of nucleotide bases in a DNA chain.
- Early sequencing methods include Maxam–Gilbert chain breakage and chain termination sequencing (Sanger method).
- Sanger sequencing is a modification of DNA replication, using modified nucleotide bases called dideoxynucleotide triphosphates (ddNTPs) that lack an OH group at the 3′ carbon.
Chain Termination (Sanger) Sequencing
- Sanger sequencing uses a primer pair to outline the target DNA, DNA polymerase enzyme, and four dNTPs, with each ddNTP labeled with a different fluorescent dye.
- Synthesis stops when a ddNTP is incorporated into the growing DNA chain, resulting in a collection of fragments of various sizes (DNA ladder).
- The fluorescently labeled DNA ladder is resolved by gel or capillary gel electrophoresis, and the sequence is read from the bottom to the top of the gel.
- Accuracy of interpretation depends on the quality of the template, sequencing reaction, and purity of the sequencing ladder.
Pyrosequencing
- Pyrosequencing is a sequencing method that relies on the generation of light (luminescence) when nucleotides are added to a growing strand of DNA.
- The procedure uses a single-stranded DNA template, sequencing primer, enzyme, and substrate, with dNTPs introduced sequentially to the reaction.
- The pyrosequencing reaction generates a pyrogram of luminescent peaks associated with the addition of complementary nucleotides.
- Pyrosequencing is less labor-intensive than Sanger sequencing and is more convenient for short sequence analyses, particularly for targeted areas around nucleotide base changes.
Next-Generation Sequencing (NGS)
- The first human genome was sequenced by chain termination (Sanger) sequencing, which took 7 years, hundreds of instruments, and billions of dollars.
- NGS technologies can sequence a human genome in a few hours, making it faster and more cost-effective.
- NGS involves massively parallel sequencing, yielding hundreds of thousands of sequences in a single run.
- The goal of NGS was to sequence the human genome for under $1,000, making it affordable for clinical analysis.
- The "1000 Genome Project" has sequenced thousands of genomes, achieving the goal of reducing the cost to under $1,000.
NGS Technologies
- Pyrosequencing, sequencing by synthesis with reversible dyes, ion conductance, and sequencing by ligation are the first mass-marketed NGS technologies.
- Pyrosequencing, reversible dye sequencing, and ion conductance sequencing are commonly used in clinical applications.
- NGS procedures begin with short DNA templates, usually less than 500 bp, and involve template preparation methods like amplification with multiple primer pairs or enzymatically digested genomic DNA.
Library Preparation
- A library is prepared by fragmentation, probe, or primer selection of specific genes or gene regions.
- Adaptors carrying PCR primer-binding sites are added to the ends of fragmented DNA.
- In a second PCR reaction, primers carry short index DNA sequences to identify each sample.
- A bar code or index is a 6- to 10-b sequence assigned to each sample and gene region in the library.
Sequencing Technologies
- Pyrosequencing uses emulsion PCR to generate libraries, which are then sequenced in a picoplate.
- Ion conductance sequencing uses a semiconductor or ion chip to detect the release of hydrogen ions during DNA synthesis.
- Reversible dye terminator sequencing uses a flow cell to amplify and sequence templates simultaneously.
Sequence Analysis
- Sequence data is collected and processed to identify variants, polymorphisms, or the sequence itself.
- The sequence quality is assessed, and the sequence is determined by comparison with stored reference sequences.
- The number of times a region is sequenced is called the coverage, which affects the accuracy of the sequence data.
Clinical Applications
- NGS has been used in genetics (including pharmacogenomics), oncology, and HLA typing.
- HLA typing has been advanced by NGS, allowing for more accurate and extensive typing.
- The Cancer Genome Atlas (TCGA) has used NGS to sequence the nucleic acid isolated from tumors of thousands of patients, leading to a better understanding of cancer and improved diagnosis and treatment.
Bioinformatics
- Bioinformatics merges biological data with information technology to analyze and interpret large amounts of data.
- The interpretation of data generated by NGS requires massive storage space and contributes to the renewal of previously stored data in organized databases.
- Software programs are used to generate reports, identify variants, and filter data based on quality, variant type, and allele frequency.
Flow Cytometry Basics
- Flow cytometry is a system that analyzes single cells in a fluid suspension based on their intrinsic light-scattering characteristics and extrinsic properties using fluorescent-labeled antibodies or probes.
- Simultaneously measures multiple cellular or bead properties using different fluorochromes.
- Each fluorochrome has a distinctive spectral pattern of absorption and emission.
Instrumentation
- Major components of a flow cytometer:
- Fluidics
- Laser light source
- Optics and photodetectors
- Data analysis and management are performed by computers.
- Fluidics: cells pass through the laser light one cell at a time, and the sample stream is constrained by the carrier stream, allowing for hydrodynamic focusing.
- Laser light source: solid-state diode lasers are typically used, and the wavelength of monochromatic light emitted by a laser dictates which fluorochromes can be used.
- Optics and photodetectors: signals generated by cells' interaction with the laser are detected by photodiodes and photomultiplier tubes.
Sample Preparation
- Samples commonly used: whole blood, bone marrow, and fluid aspirates.
- Whole blood should be collected into EDTA or heparin, and stored at room temperature before processing.
- Erythrocyte removal is required for efficient analysis of WBCs, using techniques such as density gradient centrifugation or erythrocyte lysis.
Data Acquisition and Analysis
- Data are digitized and ready for analysis after collection.
- Typically, 10,000 to 20,000 "events" are collected for each sample.
- Graphics of the data can be represented in multiple ways, including:
- Single-parameter histogram
- Bivariate histogram or dual-parameter dot plot
- Gates can be drawn around a population of interest to analyze various extrinsic and intrinsic parameters.
Clinical Applications
- Routine applications of flow cytometry:
- Nonmalignant immunophenotyping (e.g., characterization and enumeration of normal lymphocytes)
- Malignant immunophenotyping (e.g., immunophenotypic characterization of leukemias and lymphomas)
- Immunophenotyping is essential for:
- Evaluation of immunodeficiency (e.g., HIV infection, AIDS)
- Diagnosis of inherited immunodeficiency diseases (e.g., Bruton's tyrosine kinase deficiency, chronic granulomatous disease)
- Monitoring of patients with leukemias and lymphomas for "minimal residual disease"
- Determination of DNA content or ploidy status of tumor cells
- Transplant immunology (e.g., HLA typing and cross-matching)
Additional Applications
- Cytometric bead arrays:
- Can detect multiple analytes at the same time
- Theoretically, can detect 100 analytes from a single blood sample
- Examples of uses: testing for anti-nuclear antibodies, detecting fetal-maternal hemorrhage
Immunoassay Automation
- Reliable immunoassay instrumentation was first available in the early 1990s, allowing for automation of heterogeneous immunoassays even for low-level peptides such as peptide hormones.
- Over 60 different automated immunoassay analyzers are currently available, capable of performing almost all common diagnostic immunoassays, and have largely replaced manual testing in larger laboratories.
- The development of immunoassay analyzers was driven by the need to reduce manual tasks, handle large volumes of samples, and eliminate errors caused by fatigue or erroneous sampling.
- Automation decreases the likelihood of error, reduces turnaround time, and costs per test, and can provide more services with less staff.
Types of Immunoassay Analyzers
- There are two main types of immunoassay analyzers: batch analyzers and random-access analyzers.
- Batch analyzers can examine multiple samples at once, but only perform one type of analysis at a time and do not allow for stat samples to be loaded randomly.
- Random-access analyzers are modular systems that can be configured to measure numerous analytes from multiple samples, allowing for rapid processing of stat samples.
Automation in the Analytical Stage
- Automation can occur in all three stages of laboratory testing: preanalytical, analytical, and post-analytical.
- The analytical stage includes introducing a sample, adding reagent, mixing, incubating, detecting, calculating, and reporting results.
- Automatic sampling can be accomplished using peristaltic pumps, positive-liquid displacement pipettes, and stainless-steel probes with clot detectors and liquid-level sensors.
Reagent Handling and Storage
- Reagents may come ready for use or require dilution by the analyzer.
- Reagents are handled, prepared, and stored using various methods, including bar-coded reagents to reduce operator error.
- Reagents are stored in laboratory refrigerators or in reagent storage compartments within the analyzer.
Mixing and Incubation
- Analyzers use different methods for mixing, including magnetic stirring, rotation paddles, forceful dispensing, and vigorous lateral shaking.
- Timed incubation is carried out at ambient temperatures, and some analyzers have built-in incubators for temperature-controlled incubation.
Detection and Flexibility
- Detection methods include colorimetric absorption spectroscopy, fluorescence, and chemiluminescence.
- Some analyzers combine chemistry and immunoassay testing on a single platform, offering flexibility and rapid processing.
Validation of Immunoassay Analyzers
- Proper validation of new instrumentation or methodology is required before patient results can be reported with confidence.
- Validation includes verifying the manufacturer's performance specifications, meeting CLIA regulations, and complying with individual state's regulatory agency standards.
- The required verifications for a new method include accuracy, precision, analytic sensitivity, and analytic specificity, including interfering substances, reportable range, and reference intervals.
Bioinformatics and Molecular Diagnostics
- Bioinformatics is the process of collecting and analyzing complex biological data, such as genetic codes.
- Molecular diagnostics involves testing DNA and RNA, the two main types of nucleic acids.
- DNA and RNA are made up of nucleotides, which are composed of a sugar, a nitrogen base, and a phosphate group.
- The five nitrogen bases that make up the majority of nucleic acids are Adenine (A), Cytosine (C), Guanine (G), Thymine (T), and Uracil (U).
DNA Structure and Replication
- DNA is mostly double-stranded and arranged in a helix.
- DNA replication occurs through the separation of the two strands, with each strand serving as a template for a newly synthesized strand.
- This process is catalyzed by a DNA polymerase enzyme.
RNA Synthesis and Translation
- RNA synthesis is catalyzed by an RNA polymerase enzyme.
- The process of transcription occurs when genetic information flows from DNA to mRNA (messenger RNA).
- In the process of translation, the mRNA becomes a protein, which is responsible for the phenotype or genetic trait.
Mutations, Variants, and Polymorphisms
- A change in the nucleotide sequence of DNA is referred to as a mutation or variant.
- Depending on the frequency of occurrence, the sequence change could also be referred to as a polymorphism.
- Structurally, mutations, variants, and polymorphisms are all the same thing.
Electrophoresis
- Electrophoresis is a process that moves particles using an electro-current through a gas, liquid, or solid phase.
- It is useful for analyzing DNA for mutations and polymorphisms.
- Capillary electrophoresis is a more sensitive and semi-automated type of electrophoresis.
Microarray Technique
- A microarray is a tool that can detect the expression of thousands of genes simultaneously.
- It involves a slide with specific DNA probes, which act as a probe to detect gene expression.
- The process involves extracting DNA, converting it to cDNA, and then loading it onto a microarray chip.
- The chip is then scanned, and the data is analyzed to extract information on gene expression.
FISH (Fluorescence In Situ Hybridization)
- FISH is a molecular cytogenetic technique that uses fluorescent probes to detect specific DNA sequences.
- The probes are designed to bind to specific parts of a chromosome.
- The process involves denaturation of the probe and target, hybridization, and detection.
- FISH is useful for analyzing morphology, pathology, developmental biology, and karyotyping.
Fluorescence Microscopy
- Fluorescence microscopy is a powerful analytical tool that combines the magnifying properties of light microscopy with the visualization of fluorescence.
- Fluorescence occurs when a substance absorbs light at a given wavelength and emits light at another wavelength.
- The process involves using a powerful light source, specialized filters, and a means of fluorescently labeling a sample.
- Fluorescence microscopy is useful for imaging specific cell types, molecules, and structures within cells.
Fluorescence and ANA (Antinuclear Antibody)
- Fluorescence results from the property of some molecules to absorb light at one wavelength and emit it at a longer wavelength.
- The ANA test is used to evaluate a person for autoimmune disorders, such as systemic lupus erythematosus (SLE).
- The test involves mixing a person's blood sample with cells that are affixed to a slide, and then treating the slide with a fluorescent antibody reagent.
- The presence of fluorescence is noted, and the results are reported as a titer, along with a description of the particular type of fluorescent pattern seen.### Autoimmune Disorders and ANA Test
- Different patterns of antinuclear antibody (ANA) test are associated with various autoimmune disorders, including:
- Homogeneous (diffuse) pattern: associated with SLE, mixed connective tissue disease, and drug-induced lupus
- Speckled pattern: associated with SLE, Sjörgen syndrome, scleroderma, polymyositis, rheumatoid arthritis, and mixed connective tissue disease
- Nucleolar pattern: associated with scleroderma and polymyositis
- Centromere pattern (peripheral): associated with scleroderma and CREST (Calcinosis, Raynaud syndrome, Esophageal dysmotility, sclerodactyly, Telangiectasia)
ANA Test Results
- A positive ANA test result is reported as a number of units above the lab's reference number (cutoff) for the lowest possible value considered positive
- The higher the value reported, the more likely the result is a true positive
- ANA test results can be positive in people without known autoimmune disease, requiring careful evaluation in conjunction with individual signs and symptoms
- A positive ANA test result may occur before signs and symptoms of an autoimmune disease develop
Conditions Associated with Positive ANA Test
- SLE (most common condition): 95% of those with SLE have a positive ANA test result
- Drug-induced lupus: a number of medications may trigger this condition, associated with SLE symptoms
- Sjörgen syndrome: 40-70% of those with this condition have a positive ANA test result
- Scleroderma (systemic sclerosis): 60-90% of those with scleroderma have a positive ANA test result
- Other conditions: Raynaud's syndrome, arthritis, dermatomyositis, polymyositis, mixed connective tissue disease, and other autoimmune conditions
Flow Cytometry
- Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid
- It is a powerful tool for detailed analysis of complex populations in a short period of time
- Applications in hematology include:
- Immunophenotyping of various specimens, including whole blood, bone marrow, serous cavity fluids, cerebrospinal fluid, urine, and solid tissues
- Erythrocyte analysis, including detection and quantification of fetal red cells in maternal blood
- Reticulocyte counts, using fluorescent dyes that bind to residual RNA
- Immunologic monitoring of HIV-infected patients, including CD4 counts and viral load testing
- Diagnosis of leukemias and lymphomas, using immunophenotyping and antigen profiles
This quiz covers the principles and applications of gel electrophoresis in analyzing DNA mutations and polymorphisms. It explores the use of semisolid matrices to sieve nucleic acid polymers and different types of gels used in nucleic acid analysis.
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