Specimen Collection Flashcards
_____ is not as critical for most molecular testing. The quality of nucleic acids may be compromised if the specimen is ______ handled.
Viability
improperly
DNA and especially RNA will be damaged in ____ or ______. Due to the ____________, it is also important to __________ that could yield false positive results.
lysed or nonviable cells
sensitivity of molecular testing
avoid contamination
Sampling must include material from the ______infection. The time and site of collection should be _____for the likely presence of the infectious agent.
original
optimal
The quantity of target organisms, as well as clinical implications, should be taken into account when interpreting the significance of _________.
Molecular detection can reveal infective agents at levels ______ clinical significance Conversely, _____________ methods may miss detection of a variant organism.
positive results
below
highly specific molecular
Blood draws should go into the proper _________, fi one is to be used.
anticoagulant
________ swabs may be used for throat cultures.
___________swabs with plastic shafts have been recommended for collection of bacteria, viruses, and mycoplasma from mucosal surfaces.
Wooden-shafted swabs
Dacron or calcium alginate
_______are less adherent to the microorganisms and will not interfere with PCR reagents, with the exception of calcium alginate swabs with __________, which had been reported to affect PCR amplification.
Plastics
aluminum shafts
Collection methods such as____________, ______,_______ have been designed for maximum recovery of microorganisms from swabs by centrifugation.
o _________– sterile cups, screw-capped tubes, stoppered tubes, petri dishes
o ________ – calcium alginate swabs, Dacron swabs, cotton swabs, nasopharyngeal-urogenital swabs, swab transport system
o ________ – Neisseria gonorrhoeae transport systems, swab extraction tube system
o ___________ – molecular testing, N. gonorrhoeae transport systems, STAR buffer
o ___________s – Starplex anaerobic transport system (Fisher), BBL vacutainer anaerobic specimen collector
o _________ – BD cellmatics viral transport pack, BBL viral culturette
swab extraction tube system. sonication, and vortex
Sterile containers Swabs Specialty systems Proprietary systems Anaerobic transport system Viral transport systems
Isolating nucleic acids from microorganisms is similar to
isolating nucleic acids from _______ with only a few
additional considerations
human cells
Isolating nucleic acids from microorganisms is similar to isolating nucleic acids from human cells with only a few additional considerations.
o Depending on the microorganism, more________ procedures may be required. Mycobacteria and fungi, in particular, have ____ cell walls that are more difficult to lyse than those of other bacteria and parasites. __________ has a thicker cell wall than gram-negative bacteria and may require more rigorous cell lysis conditions. ______ lacks a cell wall, and so care must be taken with the sample to _________of the cells and loss of nucleic acids.
rigorous lysis thick Gram-positive bacteria Mycoplasma avoid spontaneous lysis
The concentration of organisms within the clinical sample must be considered. Samples can be ________ to concentrate the organisms within the fluid from the __________ that are often received down to volumes that are appropriate for molecular procedures.
centrifuged
milliliters of sample
Inhibitors of enzymes used in molecular analysis may
be present in __________; removal or
inactivation of inhibitors might be included in
_________ methods.
clinical specimens
specimen preparation
If RNA is to be analyzed, _________of
RNases in the sample and in all reagents and
materials that come into contact with the sample is
important.
inactivation or removal
When processing a whole blood specimen, it is
important to remove _______ and other
products of __________because they
can ___________ and thus may ___________ of nucleic acid in the sample, resulting in a _________ PCR result
hemoglobin metabolized hemoglobin inhibit DNA polymerase prevent the amplification false negative
In blood samples, white blood cells are isolated from
the red blood cells using _________ and then
lysed.
Ficoll-Hypaque
Whole blood is processed in _____________, which effectively __________ and other contaminating molecules.
automated DNA isolation systems
remove hemoglobin
__________, devoid of red blood cells, are also
used as sources of microorganism nucleic acid.
Serum and plasma
Sputum is a source of nucleic acid from organisms
that cause ______________. __________ present in sputum may inhibit DNA polymerase and thus must be removed.
respiratory tract infections
Acidic polysaccharide
___________ is treated similarly to cerebrospinal fluid; that is, the specimen is _____________the organisms and then subjected to nucleic acid isolation procedures. Inhibitors of DNA polymerase -_________________________ – have been demonstrated in urine as well.
Urine
centrifuged to concentrate
nitrate, crystals, hemoglobin, and beta-hCG
Any clinical specimen _____ used as a source of
microorganism nucleic acid for analysis.
can be
Depending on the specimen,_______________ may be necessary to allow for optimal nucleic
acid isolation, amplification, and analysis
special preparation procedures
The presence of inhibitors of DNA polymerase has been demonstrated in clinical samples.
Careful separation of nucleic acid from other molecules present in the sample will _____________.
The type of specimen used for molecular testing will also affect extraction and yield of nucleic acid.
Reagents and devices have been developed to combine collection and extraction of nucleic acid from difficult specimens.
ensure target amplification
The presence of inhibitors of DNA polymerase has been
demonstrated in clinical samples.
Careful separation of nucleic acid from other molecules
present in the sample will _____________.
The type of specimen used for molecular testing will also
affect extraction and yield of nucleic acid.
Reagents and devices have been developed to combine
collection and extraction of nucleic acid from _________
ensure target amplification
difficult specimens.
Molecular tests require _______specimen handling and processing for ______ and_____ test results. The success of a test procedure is affected by the ____,_____and ______ of specimens.
optimal
accurate; consistent
age, type, condition
___________– consequence of erroneous or misleading results caused by events that occur prior to sample analysis.
Preanalytical error
Each laboratory will have requirements for specimen handling, but __________ apply to all specimen collection.
The condition of the specimen and the chain of custody is reviewed on__________in the laboratory
general policies
receipt
If a specimen shows evidence of tampering or is hemolyzed, degraded, clotted or otherwise compromised, the technologist must ____________.
notify the superviso
No specimen is accepted___________ and
identification on the specimen tube or container, nor is a
specimen accepted if the labeling on the ______________ that on the accompanying requisition.
without proper labeling
specimen does not match
If a specimen is unacceptable, the ____________ of
the specimen is recorded in the _________ or
laboratory quality assurance records.
disposal or retention
patient report
If not processed immediately, specimens are maintained
in ________________under the appropriate
conditions for the analyte being tested.
secure areas with limited access
For routine specimens tested by ___________,
the entire specimen is often not used. If more than one
test is to be performed on the same specimen, ___________ of specimens is carefully avoided. This can
most likely occur from ________ carryover.
amplification methods
cross-contamination
pipetting
An aliquot removed from a specimen is __________ to
the original tube or vessel. Molecular genetic tests may
require dedicated specimens not shared with other tests.
never returned
________ inhibits enzyme activity. Specimens should be
inspected for visual signs of _________. ________ and
_______ are removed effectively in most DNA and RNA
isolation procedures; however, if white blood cell lysis has
also occurred, DNA or RNA yield will be reduced. This
could result in _______ results in qualitative testing
or _______________in quantitative analyses.
Hemoglobin hemolysis Hemoglobin and coagulants false-negative inaccurate measurements
______ and _________amplification are routinely performed on
paraffin-embedded tissue samples.
______________, requiring large fragments of
DNA, are less likely to work consistently with fixed tissues.
PCR and RT-PCR
Southern or northern blot
Collection Tubes for Molecular Testing:
1. \_\_\_\_\_\_\_\_\_\_\_\_ – chemistry, serum, viral antibody studies 2. \_\_\_\_\_\_\_\_\_\_\_\_\_\_ – immunology, virology studies 3. \_\_\_\_\_\_\_\_\_\_\_\_ – cytogenetic studies, molecular studies 4. \_\_\_\_\_\_\_\_\_\_\_\_ – virology, molecular biology studies 5. \_\_\_\_\_\_\_\_\_\_\_\_\_\_ – molecular biology studies
(Red) No additive (Green) Sodium heparin freeze dried (Brown) Sodium heparin (Lavender) Tripotassium EDTA (Yellow) Acid citrate dextrose solution
___________– recommended by the CDC for
handling potentially infectious specimens. All specimens
are potentially infectious, so they should all be handled
with standard precautions using proper personal
protective equipment to prevent disease transmission.
Standard precautions, including ______ as PPE,
are used by the molecular laboratory technologist who
has no direct contact with patients.
Standard precautions
____________ precautions – include respirators; used with airborne or contact-transmissible agents.
Transmission-based
_______________ – for direct patient care where there is the potential for direct exposure to infectious agents on or form the patient.
Contact precautions
_________ are also important to protect nucleic acids from
nuclease degradation and are absolutely required for
handling of RNA.
Gloves
_____ is less susceptible to degradation from contaminating
DNases.
DNA
Repeated handling of samples _________ will
adversely affect the integrity of the DNA over time.
without gloves
Standards and controls that are handled _________ are
the most likely to be affected. Having separate areas for
DNA and RNA isolation is ______.
repeatedly
recommended
Change in assay response with corresponding change
in analyte.
Ø Ex: All positive reference standards tested positive
with the new assay. The analytical sensitivity of the
assay is 100%.
Analytic sensitivity
Ø Ability of test result to predict a clinical condition.
Ex: Of 100 patients with a gene mutation, 95 have a
disease state, a clinical sensitivity of 95%.
Clinical sensitivity
Ø Least detectable presence of the analyte
Ex: The t(14;18) translocation test can detect 1
translocated cell in 10, 000 normal cells, a detection
limit of 0.01%.
Detection limit or Limit of detection
Ø Ability to detect only the analyte and not nonspecific
targets
Ex: The invader assay for factor V Leiden successfully
detected mutations in 18 positive specimens while
yielding negative results for 30 normal specimens; no
false positives.
Analytic specificity
Ø Disease-associated results only in patients who
actually have the disease conditions
Ex: Of 100 normal specimens, 1 displayed a gene
mutation; 1 false positive, a clinical specificity of 99%.
Clinical specificity
Ø Agreement between independent test results.
Ø Ex: A quantitative method yields 99 results less than
1 standard deviation apart in 100 runs, a precision of
99%.
Precision
Ø Consistency of test results produced from the same
procedure.
Ø Ex: A qualitative method yields 100 positive results
when performed in 10 independent laboratories, a
reproducibility of 100%.
Reproducibility
Ø The range within which a specimen may be measured
directly without dilution or concentration.
Analyte measurement range
Ø The range within which test results are considered to
be valid with or without dilution.
Reportable range
Ø Expected analyte frequency or levels from a
population of individuals.
Ø Ex: The reference range for prostatic specific antigen
is 0-4 ng/mL.
Reference range
Ø Production of correct results.
Ø Ex: Of 100 specimens with mutations in the HCM
gene, 99 are detected by sequencing, with no
mutations detected in normal specimens.
Analytic accuracy
Ø Quantitative correlation between test result and actual amount of analyte.
Ø Ex: A graph of test procedure results versus input
analyte yields a straight line.
Linearity
_________– establishment of conditions for
instrument response/method result association with
the true value of quantitative analyte measurements.
Calibration
__________ – samples of known
amounts/concentrations of molecules of the same
type measured in the assay, such as an admixture of
PCR products in known proportion or an RNA
transcript calibrator in a matrix of normal RNA. The
source, quality, and preparation of the calibrators
should be documented.
Calibrators
______________– performed if calibration does not
meet the required standards of linearity or accuracy.
Re-calibration
_____________ – may be required upon changes of or major repairs to instruments or changes in reagent lots that might affect test performance or when quality control indicates shifts or unacceptable errors in test control results. Verification should demonstrate the continued linearity of correlation
between the calibrator values and test results.
Calibration verification
These are samples of known type or amount that are
treated like and run with patient specimens.
controls
Interpretation of test results always includes
inspection of ______and ______ to verify
acceptable test performance.
controls and standards
The controls and standard curve should cover the
___________levels or results of the method.
__________are continually monitored to spot
trends or spikes outside of tolerance limits.
critical detection
Control results
___________– defines lower limit of detection for more meaningful interpretation of negative results. For qualitative tests, along with positive and negative controls
Sensitivity control
_______________– a target should always amplify. It distinguishes true-negative amplification results from false negatives resulting from amplification failure. For nucleic acid amplificationtechniques. In quantitative methods, high-positive,
low-positive, and negative controls are included with
each run. The high and low levels should be similar to
critical points in the assay, such as the lowest
detectable level of analyte.
Amplification control
____________ – or dilution series of analyte levels
encompassing the levels expected from the patient
specimens. Real-time PCR methods automatically
determine analyte levels that require measurement
of this curve or dilution series.
Standard curve
____________ – run in the same reaction mix as the
test specimen; in methods requiring detection of a
target-specific product or relative amounts of target.
Internal control
____________________ should be
included in each run of patient samples
Positive, sensitivity, and negative controls
For tests with multiple targets, controls can be
systematically rotated for different targets in each run.
• Controls for quantitative tests should reflect the __________ levels of test results.
• _________ or acceptability levels of control values should
be established based on the validated precision and
reproducibility of the assay.
clinically critical
Tolerance
Monitoring can be performed using ___________ or
modified LJ plots or cusum plots, and tolerance limits can
be expressed as __________.
If the control results violate the set limits, then corrective
action such as _____________has to be taken and documented.
Levey-Jennings
Westgard rules
repeating the run with replacement controls
If patient specimen availability precludes repeating a run,
___________ into the nature of the ___________
and ___________ result ____ allow acceptance
of the sample results without repeating.
further investigation
control failure
its effect on the patient
may
Comparison of test values with previous results or
historical averages may _______________ in
the test results
serve to increase confidence
Monitored controls and conditions are reviewed ________
or at determined intervals to document outliers or
systemic error.
monthly
Periodic review and documentation of test results are
______ for all clinical testing, including molecular tests
required
Critical values that require physician notification are
established by ________ and confirmed by _______.
validation
monitoring
Assay levels that distinguish positive from negative
results, cut-off values or cut points, must be _______
and verified at regular intervals
well defined
must be well defined and verified at regular intervals.
Also includes documentation and maintenance of
laboratory procedures and methods. These procedures
are frequently updated with events ranging from major
advances in instrument technology to changes in reagent
availability.
Test results may show _________ with other laboratory
findings, with clinical observations, or with the
laboratory’s own preliminary results.
discrepancies
____________ are documented along with any corrective
action taken, if necessary.
Discrepancies
Due to the nature of molecular pathology testing,
discrepancies may be explained by the technical aspect of
the test.
o Increased sensitivity
o High resolution
o Varied methodologies used
HAZARDOUS MATERIAL CLASSIFICATION
1
2
3
W
REACTIVITY
FIRE HAZARD
HEALTH HAZARD
SPECIFIC HAZARD
SPECIFIC HAZARDS: W
OXIDIZER ACID ALKALI CORROSIVE USE NO WATER RADIATION
REACTIVITY
4 3 2 1 0
May deteriorate Shock and heat may deteriorate Violent chemical change Unstable if heated stable
FIRE HAZARD
4 3 2 1 0
Below 73*F Below 100*F Below 200*F Above 200*F Will not burn
HEALTH HAZARD
4 3 2 1 0
Deadly Extreme Danger Hazardous Slightly hazardous Normal material
