Molecular Techniques & POCT Flashcards
assays that target nucleic acid instead of protein, are the
latest development in clinical laboratory testing.
Molecular techniques,
requires the release of DNA or RNA from the cell
followed by its purification and quantitation.
Nucleic acid extraction
is a short strand of DNA or RNA of a known sequence that
is well characterized and complementary for the base sequence on the test target.
nucleic acid probe
based on the reversible complementary base
pairing of nucleotides.
principle of hybridization
Southern blot
Northern blot
In situ hybridization
Restriction fragment length polymorphism
Probe Techniques-Unamplified:
PCR
RT-PCR
Transcription based amplification systems
Transcription-mediated amplification
Nucleic acid sequence-based amplification
Strand displacement assay
Probe Techniques-Target Amplification
Probe Techniques-Probe Amplification
Ligase chain reaction
Probe Techniques-Signal Amplification
Branched DNA assay
are the simplest types of solid
support hybridization assays.
Dot blot and sandwich hybridization assays
clinical samples are applied
directly to a membrane surface. The membrane is heated to denature or separate
DNA strands, and then, labeled probes are added.
dot blot assay,
modification of the dot blot procedure. It was
designed to overcome some of the background problems associated with the use
of unpurified samples.
Sandwich hybridization
In this method, DNA is extracted from a sample using a
phenolic reagent and then enzymatically digested using restriction endonucleases
to produce DNA fragments.
Southern blot.
is a technique that
evaluates differences in genomic DNA sequences. This technique can help
establish identity or nonidentity in forensic or paternity testing or to identify a
gene associated with a disease.
Restriction fragment length polymorphism (RFLP)
RNA is extracted, digested, electrophoresed, blotted, and
finally probed.
Northern blot
for the
detection of human papillomavirus (HPV) is a solution-phase hybridization
assay that uses an antibody specific for DNA/RNA hybrids to “capture” and
detect the hybrids that are formed during the solution hybridization of HPV
DNA in the sample with an unlabeled RNA probe.
Digene Hybrid Capture 2 assay
In this type of
setting, both the target nucleic acid and the probe are free to interact in a reaction
mixture, resulting in increased sensitivity compared with that of solid support
hybridization.
solution hybridization
is considered the “gold standard” for many molecular
applications from mutation detection to genotyping, but it requires proper
methodology and interpretation to prevent misinterpretation.
DNA sequencing
is a relatively new technique for a short to moderate
sequence analysis that is based on the release of pyrophosphate during DNA
synthesis as each dNTP (deoxyribonucleotide triphosphate) is incorporated with
elongation of DNA.
Pyrosequencing
are very small devices used to examine DNA, RNA, and
other substances.
Biochips/microarrays
is an amplified
hybridization technique that enzymatically synthesizes millions of identical
copies of the target DNA to increase the analytic sensitivity.
PCR
When the target is microbial RNA or mRNA, the RNA must be
enzymatically converted to DNA by reverse transcriptase; the product, cDNA,
can then be analyzed by PCR.
RT-PCR
allows for direct measurement of amplicon accumulation during the exponential
phase of the reaction.
“Real-time” RT-PCR
defined as the value at which the sample fluorescence crosses the threshold.
The threshold cycle (CT)
A thermostable enzyme; 5’-3’ polymerase activity
Taq polymerase
Needed for polymerase & nuclease activities of Taq polymerase
Magnesium salts
is defined as the temperature at which 50% of the
DNA is double stranded and 50% is single stranded.
DNA melting temperature (Tm)
detects target RNA and
involves continuous isothermic cycles of reverse transcription.
self-sustained sequence replication or
transcription-based amplification system (TAS)
The principle of the reaction was a two-
step process that involved generation of cDNA from the target RNA followed by reverse transcription of the cDNA template into multiple copies of RNA.
Transcription-based amplification
One set of
primers incorporates a specific restriction enzyme site that is later attacked by an
endonuclease. The resulting “nick” created in only one strand by the restriction
enzyme allows for displacement of the amplified strands that then, in turn, serve
as targets for further amplification and nick digestion.
Strand displacement amplification (SDA)
is a probe amplification technique
that uses two pairs of labeled probes that are complementary for two, short-target
DNA sequences in close proximity.
ligase chain reaction (LCR)
are designed to increase the signal strength by
increasing the concentration of the label.
Signal Amplification
are useful in identifying microorganisms in a patient
specimen or confirming an organism isolated in culture.
Nucleic acid probes
defined as “those analytical patient-testing
activities provided within the institution, but performed outside the physical
facilities of the clinical laboratories.”
Point-of-care testing (POCT)
“near-patient testing,” “extra-laboratory analyses,” “ancillary testing,” “bedside testing,” “physician's office testing,” “alternative site testing.”
POCT
The major advantage of POCT
faster delivery of results.
CLIA certificate: Issued to a laboratory to perform only waived tests
Certificate of Waiver
CLIA certificate: Issued to a laboratory that enables the entity to conduct moderate- or high-complexity lab testing or both
Certificate of Registration
CLIA certificate: Issued to a lab in which a physician, midlevel practitioner, or dentist performs no tests other than the microscopy procedures
Certificate for PPMPs
CLIA certificate: Issued to a lab after an inspection that finds the lab to be in compliance with all applicable CLIA reqs
Certificate of Compliance
CLIA certificate: Issued to a lab on the basis of the lab’s accreditation by an accreditation organization approved by the Health Care Finance Admin
Certificate of Accreditation
3 categories in POCT complexity
Waived tests
Moderate-complexity tests
High complexity tests
of tests defined by CLIA, such as
dipstick tests, urine pregnancy tests, and blood glucose monitoring devices,
which are subject to the lowest level of regulation and are cleared by the FDA
for home uses.
Waived tests
4th category of POCT: These tests
involve the use of a microscope, limited to bright-field or phase-contrast
microscopy. Generally, the specimens are labile and cannot survive transport to a
clinical laboratory.
provider-performed microscopy procedures,
or PPMPs
TRUE/FALSE: There are usually no QC materials available for PPMP;
however, the individual performing PPMP endures the certification process and
participates in proficiency testing.
TRUE
scientific
oversight of the POCT
technical consultant
required to provide
clinical and medical advice.
clinical consultant
monitors day-to-day
activities of testing personnel, and it is the responsibility of the ____ to
coordinate POC patient testing and facilitate compliance with procedures and
policies and regulatory requirements.
POCT coordinator (POCC)
ensures the
electronics of the device are performing as expected or, if a manual test, the
integrity of the specific test system.
Internal QC
onboard QC, internal checks, electronic QC, or intelligent QC
Internal QC
The greatest source of error in POCT
Preanalytical error
Preventing postanalytic errors in reporting can be achieved more reliably
with _______.
connectivity
Reflectance Electrochemistry, electrical impedance Light scattering/optical motion Immunoturbidimetry Lateral flow, flow-through, or solid phase immunoassays Spectrophotometry, multiwavelength spectrophotometry Fluorescence, time-resolved fluorescence Polymerase chain reaction
analytical principles
used in a laboratory have also been implemented in POCT devices
If QC has not
been performed as required and is unsatisfactory, the instrument does not allow
patient testing until corrective action has taken place.
QC lockout
