Term
| Specimen Labeling and Test Requisitions |
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Definition
Specimen must have labels with patient identifiers matching the TRF. Samples with dissimilar identifiers on both are unacceptable and must be recorded as such.
TRFs must include: specimen type, ordered test, collection date, OD contact.
Molecular genetics/parentage tests might require patient consent, ethnicity, photo ID of patient, transfusion history, pedigree.
If not processed immediately, samples must be kept in a limited access area. |
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Term
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Definition
| Molecular tests use very little sample material, so leftover material may be kept for repeat testing. Cross contamination of samples must be avoided, especially from pipetting carryover. Aliquots removed from a specimen can never be returned to the original container. |
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Term
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Definition
| As hemoglobin inhibits enzyme activity, hemolysis should be monitored for in samples. Most DNA/RNA isolated procedures remove hemoglobin/anticoagulants. If white blood cells lyse, nucleic acid yield will be low, causing false negatives or inaccurate measurements. Buffers can sequester anticoagulants or hemoglobin for rapid isolation and prevent changes in samples caused by storage. |
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Term
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Definition
Tissue analysis is best from fresh or frozen samples, so should be snap frozen immediately if not processed upon arrival.
Fixed tissues yield low-quality nucleic acid, but may be adequate for PCR and RT-PCR, but not good for blots. |
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Term
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Definition
| An anticoagulant that can inhibit enzymes used in molecular analysis (polymerases, RT). This compromises amplification of long DNA fragments more than short ones, so may be acceptable for some tests. Signal amplification techniques are less susceptible. |
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Term
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Definition
| Found in royal-blue-capped tubes used for trace element studies. Inhibits enzymes, should be avoided. |
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Term
| Acid Citrate Dextrose Tubes |
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Definition
| Yellow. Used for molecular biology. |
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Term
| No-Additive Collection Tubes |
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Definition
| Red caps. Used in chemistry, serum, viral, and antibody studies. |
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Term
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Definition
Green caps - used for freeze-dried immunology and virology.
Brown caps - used for cytogenetic and molecular studies. |
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Term
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Definition
7.5-15% solution.
Lavendar caps. Used for virology and molecular biology. |
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Term
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Definition
Contain anticoagulants and a gel matrix that sequesters granulocytes and some lymphocytes from erythrocytes when centrifuged. These are used for HIV, HCV tests, bone marrow engraftment testig, and white cell collection for molecular genetics.
The caps have a black "tiger" pattern over the standard anticoagulant color. |
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Term
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Definition
Samples must be handled consistently to ensure accuracy of serial analysis (e.g., viral load testing).
Tubes containing proprietary RNA stabilizing agents can be used to maintain RNA integrity until isolation. Standard tubes can be used if WBCs are lysed in trizol/triReagent and stored at -70 C to stabilize RNA. This should be immediate.
Impregnated paper matrices can be used for immediate stabilization and room temperature storage, but can only be used for amplification studies and are affected by humidity. |
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Term
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Definition
| Isolated DNA can be held for a few months or 1 year in the fridge. Purified DNA is good for 10 years at -20 - -70 C in the correct tubes. If DNA is clean and will be used several times, it should be stored in the fridge to avoid freeze thaw, which can shear DNA. Don't use frost0free freezers for this reason. |
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Term
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Definition
| Viral DNA in whole blood should be stored room temperature (22-25 C) otherwise refrigeration can degranulate neutrophils and release enzymes that affect viral particles. RNA in EDTA tubes can be stored at 4 C fine. Blood or bone marrow for RNA testing should be processed within 2-4 hours. If shipped, they must be shipped overnight at room temp or kept on cold packs. Tissues need dry ice. |
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Term
| DNA Storage in Whole Blood, Buffy Coat, Bone Marrow, Fluids |
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Definition
24 hrs --- 22-25 C
75 hrs --- 2-8 C
1 year --- -20 C
>1 year --- -70 C |
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Term
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Definition
No --- 22-25 C
24 hrs --- 2-8 C
2 weeks --- -20 C
2 years --- -70 C |
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Term
| DNA Storage in Microorganisms in Culture |
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Definition
24 hrs --- 22-25 C
72 hrs --- 2-8 C
2-4 weeks --- -20 C
> 1 year --- -70 C |
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Term
| DNA Storage in Cell Lysates in GITC |
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Definition
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Term
| RNA Storage in Whole Blood, Bone Marrow, Buffy Coat, Fluids |
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Definition
No --- 22-25 C
2-4 hrs --- 2-8 C
2-4 weeks --- -20 C
>1 year --- -70 C |
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Term
| RNA Storage in RNA Protection Tubes |
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Definition
5 days - 22-25 C
7 days --- 2-8 C
2-4 weeks --- -20 C
7 months --- -70 C |
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Term
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Definition
No --- 22-25 C
No --- 2-8 C
No --- -20 C
2 years --- -70 C
2 years --- -140 C (Nitrogen vapor) |
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Term
| RNA Storage in Cell Lysate in GITC |
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Definition
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Term
| RNA Storage in Cell Lysates in Ambion |
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Definition
1 week --- 22-25 C
1 month --- 2-8 C
>1 year --- -20 C |
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Term
| RNA Storage in Microorganisms in Culture |
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Definition
24 hrs --- 22-25 C
72 hrs --- 2-8 C
2-4 weeks - -20 C
>1 year --- -70 C |
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Term
| Isolated DNA Storage Requirements |
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Definition
In TE Buffer/DNase-free water --- 22-25 C, 4 months
Freeze-Dried or on collection paper --- 22-25 C, >15 years
TE/DNase-free water --- 2-8 C, 1-3 years
TE/DNase-free water --- -20 C, 7 years
TE/DNase-free water --- -70 C, >7 years |
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Term
| Isolated RNA Storage Requirements |
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Definition
TE/RNAse-free (DEPC) water --- 22-25 C, Never
TE/DEPC water --- 2-8 C, Never
TE/DEPC water --- -20 C, 1 month
RNA Storage Solution (Ambion) --- -20 C, >1 month
Ethanol --- -20 C, >6 months
TE/DEPC water --- -70 C, 30 days
Ethanol --- -70 C, >6 months |
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Term
| Clinical Sensitivity Equation |
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Definition
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Term
| Clinical Specificity Equation |
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Definition
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Term
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Definition
| [TN+TP]/[TN+TP+FN+FP] x 100 |
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Term
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Definition
Ability of a test result to accurately predict a clinical condition.
(95 of 100 patients with the mutation have the clinical condition, 95% sensitivity.) |
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Term
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Definition
Change in assay response in proportion with change in analyte. The ability of the test to detect the presence of the analyte.
In quantitative assays, defined by differences in quantitative response with quantitative differences in the analyte. If the numerical result will be reported qualitatively (e.g., positive), cut point quantities must be established. |
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Term
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Definition
| Ability to detect the target only, no other targets. |
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Term
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Definition
Disease-associated results are reported only for patients who have the actual condition.
(1 out of 100 negative samples was resulted as a false positive, specificity of 99%) |
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Term
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Definition
Lower limit the analyte can be detected at consistently. Is equal to the analytical sensitivity in qualitative assays (e.g., positive/negative, no quantifiers).
(1 translocated cell out of 10.000 can be detected, detection limit of 0.01%) |
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Term
| Analyte Measurement Range |
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Definition
| Range within which analytes can be measured as is, no dilution or concentration required. |
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Term
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Definition
| Range within which the test results can be considered valid (with or without dilution). |
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Term
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Definition
| Expected frequency or level of analyte in a population. |
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Term
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Definition
| Production of correct results. |
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Term
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Definition
| Quantitate correlation between test result and the actual amount of the analyte. |
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Term
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Definition
| A molecular test accepted as safe and effective based on the manufacturers data. The manufacturer must conduct tests to show the method will perform as claimed and doesn't present undue risk. The test can be sold after review and approval by the FDA. |
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Term
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Definition
| Them manufacturer has data showing the method gives results similar to tests on the market. Comparison results must be presented to the FDA as a "premarket verification". The FDA will review it and determine that the test is equivalent. |
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Term
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Definition
- Primer/probe sequences, purification, labeling
- Copy of reagent prep form
- Description of formulas and ranges used for results
- Melting temperatures
-Justification for result rejection
SOPS must be reviewed yearly and kept for two years after retirement. |
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Term
| Qualitative Test Controls |
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Definition
- Positive control
- Negative control
- Sensitivity control - defines lower limits of analyte detection to make negative results more meaningful. |
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Term
| Quantitative Test Controls |
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Definition
- High-positive
- Low positive - similar to lowest detectable level of analyte
- Negative |
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Term
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Definition
| A dilution series of analyte levels expected to be in specimens. The curve can be run simultaneously as specimens or may by previously determined and loaded into software. Manual calculations can be done via linear regression. |
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Term
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Definition
Used when a specific target is detected, or the relative levels of the target. Housekeeping RNA genes are frequently used for infectious agent quantification or tumor-specific transcript expression.
Can distinguish dales negative results from failures. Can serve as a normalization base.
Centromeric probes in FISH assays. |
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Term
| Refrigerators and Freezers |
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Definition
Max/min temperature must be monitored daily. Opening and closing of door must be taken into account. Temperatures out of range must be checked regularly before they either return to an acceptable range or necessitate further action.
Contaminated specimens and expired reagents should be disposed of immediately or sequestered away from in-use reagents. |
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Term
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Definition
| Monitored annually with a tachometer to determine actual speed vs. set speed. This should be posted on the instrument. |
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Term
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Definition
Temperature control in the block can decline over time. Temperature control should be checked periodically. Type K thermocouples (flexible probe thermometers) can be used to check representative wells.
Test reactions can be run in specific wells to demonstrate successful amplification.
Computer programs can track temperature, ramping, etc. Nonblock cyclers are monitored using capillary probes. qPCR systems need their detection system checked. |
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Term
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Definition
Electrophoresis power supplies are checked annually to verify voltage and current. Leads and connectors to baths should be kept free of precipitate by not leaving buffer in between runs.
Capillary systems need to be cleaned of buffer and gel delivery channels must be cleaned twice a month.
Capillaries have a life span in number of uses, after which they should be discarded.
Temperature is monitored. |
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Term
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Definition
Autoradiogram equipment is maintained with fixing and developing solutions free of debris or sediment.
Cameras are securely mounted and optimally adjusted to record info and kept free of dirt, shadows, other artifacts.
Fluorometric devices may need background measurements. Light source lifespan should be tracked. |
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Term
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Definition
| Checked annually or more frequently. Supplied filters are used to scan through the range of wavelengths. Measurements at 260, 280 320 nm are checked for linearity of concentration measurements. |
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Term
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Definition
Proper air flow is monitored annually, usually by facility technicians.
Functional characteristics should be monitored to identify trends that may develop. Tolerance levels should be set. |
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Term
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Definition
| Accuracy should be checked before initial use and every 6 months after. Gravimetric systems where pipetted volumes are weighed and the weight is converted to volume via density are used to measure accuracy. Standard deviation (coefficience of variance) is used to determine imprecision. |
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Term
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Definition
Adjustment of an instrument/test system output to an analyte of known concentration.
Calibration verification is the testing of samples of known concentrations throughout the reportable range. If this fails, recalibration is required.
Calibration is required every 6 months, when reagents/software/instrumentation is changed (required by CLIA). Also required if a PT sample fails or there's a major instrument malfunction. |
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Term
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Definition
Previously resulted specimens, reference standards, and PT material can be used in calibrations. The calibrator used must have been independently assessed for actual measurement. Calibrator results are expressed as a range, and levels should cover low, medium, and high points of the analyte.
Calibrators should be in the same matrix as the test specimens (e.g., plasma). These are different than quality controls. |
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Term
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Definition
| May be updated to reflect discovery of new markers. Probe sequence should be documented and updated whenever new probes are added. qPCR probes are supplied in solution and may need to be diluted to create a working stock. When new dilutions are performed, the resulting working stock are qualified as new lots. |
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Term
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Definition
| Usually supplied lyophilized (freeze-dried) from manufacturers, along with info on quality, purification method, molecular weight, and micrograms of primers. This is used to rehydrate primers to a stock concentration which can be diluted into working stocks. Working stocks can combine forward and reverse primers. |
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Term
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Definition
| Written lab protocols should include instructions for prep or primers, probes, working stocks. Should also include primer sequence, binding site, and length of amplicon. Factors that will affect primer binding, such as polymorphisms or translocation breakpoints, should be documented as well as their expected frequency in the population. |
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Term
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Definition
- Hybridization probes in solution are treated as working stock and diluted directly in hybridization buffer.
- RNA probes are stored in RNase-free conditions.
- Stock solutions are qualified in parallel analysis with old lots. |
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Term
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Definition
- Type of probe (e.g., cDNA, DNA, RNA, riboprobe, etc.)
- Probe sequence origin
- Probe sequence
- Known polymorphisms, restriction enzyme resistant sites, cross-hybridizing bands
- Recombination frequencies and positions, for linkage studies
- Location of target gene and known alleles, for inheritance disease tests |
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Term
| Analyte-Specific Reagents |
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Definition
ASRs. Probes, primers, antibodies. Detect a target. The active part of "home-brew" tests.
- Class I - most ASRs used in molecular labs. Not subject to special controls by the FDA. Test performance is determined in the validation of the LDT.
- Class II and III - used by blood banks to screen for infectious diseases and some contagious diseases. |
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Term
| In Vitro Diagnostic Reagent Sets |
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Definition
IVD. Used to diagnose diseases or conditions. Usually include reagents for collection, preparation, and examination of samples. Must be used according to manufacturer's protocol.
Classified from class I (low risk) to class III (high risk) based on performance risk and surveillance requirements. Performance of reagents must be verified by the lab using them. If the protocol is changed it must be validated to show equivalent performance. |
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Term
| In Vitro Analytical Test Reagents |
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Definition
| IVAT. Only the analytical equivalency of the test is monitored by the FDA, not the clinical utility. The lab is responsible for medical claims. Used to accommodate promising high complexity technologies which would have a long clearance process. |
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Term
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Definition
Not to be used for diagnostic purposes. User to gather data than can advance products.
Research Use Only (RUO) - not for use on patient samples.
Investigational Use Only (IUO) - can be used on patient samples with prior review and consent (e.g., clinical trials). |
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Term
| Radioactive Chemical Documentation |
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Definition
The Nuclear Regulatory Commission requires that a radiation safety manual is maintained that describes proper handling of radioactive chemicals in routine and extreme situations.
Signs developed by OSHA must be used to warn about hazards, radiation tags, and accident prevention. |
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Term
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Definition
Radioactive methods only occur in designated work spaces. Surfaces are covered with absorbent paper, drip trays. Fume hoods are used to potentially volatile reagents. Containers/equipment are labeled with "caution radioactive material" signs (freezers, cabinets).
Work spaces are decontaminated daily and checked for contamination monthly via swipe test or Geiger Counter. |
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Term
| Radioactive Chemical Storage and Disposal |
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Definition
Some isotopes with short half-lives can be stored for roughly 7 half-lives. They must be checked for residual emissions before being discarded in normal waste.
Storage freezers and cabinets must have signs. Radioactive waste has its won containers separate from regular waste. |
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Term
| Radioactive Chemical Handling |
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Definition
Lab workers must receive special safety, handling, and disposal training.
Shipments must be inspected and monitored.
Gloves, goggles, and lab coats required.
When handling >1.0mCi of radioactive reagents, radiation badges must be worn.
Exposure increases with increasing proximity to radioactive reagents.
Some isotopes require acrylic shielding in work, storage, waste areas. |
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Term
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Definition
A reference source (e.g, CAP) supplies samples to independent labs to assess lab personnel competency and the accuracy/reproducibility of the test itself.
CAP supplies PT samples for molecular genetics, molecular oncology, and molecular microbiology.
PT must be done twice a year (every 6 months). PT samples must be treated as patient samples. All errors and corrective actions on these samples must be documented. |
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Term
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Definition
| CAP does not offer PT samples for all tests, especially ones in small/new labs. These abs must develop their own PT protocol, either through a split specimen exchange with other labs or specimens measured by independent means (chart reviews). Interlaboratory testing preferred. |
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Term
| Electrophoresis Results Documentation |
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Definition
Electropherograms/gel images used for results should be clear and clean, with no doubt as to the result. Bands/peaks should be defined, no background, cross-hybridization, or other artifacts.
Controls should be clear, consistent, and accurately reflect the analyte.
Molecular weight ladders should more than cover the range of expected sizes. |
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Term
| Patient Result Documentation |
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Definition
| Patient results should include assay conditions and reagent lot numbers and possibly technologists. Quality of extracted nucleic acids must also be included. DNA quantity/quality can be shown via spectrophotometry, fluorometry, gel photographs. RNA run on Northern blots or RT-PCR can be qualified by monitoring calibrators (house-keeping genes, rRNA expression). |
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Term
| Restriction Enzyme and Hybridization Results Documentation |
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Definition
Efficiency of restriction enzyme must be shown to be complete via control targets on gel images. DNA cut with DNase is monitored similarly.
In hybridization arrays labeling is documented by measurement of specific activity (signal per ng or nucleic acid).
For blots, patient ID, gel lane, probe target/type must be recorded. Buffer conditions and lot numbers are a plus. |
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Term
| In Situ Results Documentation |
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Definition
| Images of sample cells are correlated with stained tissue sections. One normal cell image and two abnormal ones are required. Other images from the sample are also kept and labeled with patient ID, target analyte, sample numbers, run identifiers, and date. |
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Term
| Importance of Documentation |
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Definition
| Raw data are included and kept with the final report and clinical interpretation. Results may differ across labs, especially for sensitive assays such as amplification. Discrepancies can be investigated using raw data (investigations must also be documented). |
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Term
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Definition
Peaks/bands for nucleotides must be unquestionable. An acceptable amount of background must be shown. Defined limits of fluorescence should be set so that homozygous peaks are twice the height of the two peaks for heterozygous ones. Both strands should be sequenced and nucleotide sequence (not including the amplification primers) around the target must be accurately called.
Mutations should be compared against published guidelines to determine significance. |
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Term
| Result Report Requirements |
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Definition
- Unambiguous terms clear to non-scientists must be used
- Method/kit used
- What was tested (locus, organism, etc.)
- Analytical and clinical interpretations of raw data
- Limits of detection/resistance/penetrance
- False positive/negative likelihoods
- Mutation rates for genes
- Paternity index, prior probability, reference population |
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Term
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Definition
Lab director/other expert must review the data and give a clinical interpretation before signing off on the report (electronically or manually). Director must review results before they're resulted. Any technical complexities that might influence the report are included.
Tests using class I ASRs must include a disclaimer except for reagents sold with equipment as kits, reagents sold with instructions, or FDA-approved tests. |
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