Term
| Why is hybrid capture assay considered signal amplification? |
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Definition
| DNA is isolate, bound to a probe and then a label is bound to the probe hybrid molecule, the signal is measured in RLUs |
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Term
| What are RLUs converted into? |
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Definition
| ratio with the appropriate positive cutoff value (RLU/CO) |
|
|
Term
| What does RLU > 1 indicate? |
|
Definition
|
|
Term
|
Definition
| digene diagnostics hybrid capture for HPV |
|
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Term
| What was the first molcular test with widespread use in medical practice for detection of HPV? |
|
Definition
|
|
Term
| What are the low risk HPV? |
|
Definition
| 6 and 11 associated with genital warts |
|
|
Term
| what are the high risk HPV? |
|
Definition
| 16- squamous cell carcinoma and 18-adenocarcinoma |
|
|
Term
| What is the HPV DNA testing used as an adjunct to Pap testing called? |
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Definition
|
|
Term
| When is the HPV reflex performed? |
|
Definition
| ASC-US results and in conjunction to pap test in women > 30 years old |
|
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Term
| What is a disadvantage to hc2? |
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Definition
| is has a lower specificity than the pap test |
|
|
Term
| what is an advantage to hc2? |
|
Definition
| lets the physician know is the virus is present |
|
|
Term
| What is careHPV and where is it used |
|
Definition
| simplified version of hc2 used in third world countries as a primary screening test, where prevalence of cervical cancer is high |
|
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Term
| Where is there a high prevalence of cervical cancer in women ages 15-44? |
|
Definition
| Eastern Asia, Southern Asia, and Melanesia |
|
|
Term
| What is the 2nd most common cancer among worldwide? |
|
Definition
|
|
Term
| where do most of the cases occur? |
|
Definition
|
|
Term
| What is the general procedure of the hybrid cancer? |
|
Definition
| samples of lysed and target DNA is released from cells, DNA is denatured into single strands, is HPV is present then the HPV DNA will hybridize to the single-stranded RNA probes that are HPV specific |
|
|
Term
| What captures the DNA/RNA hybrids? |
|
Definition
| a microplate containing immobilized antibodies which are specific for RNA-DNA hybrids |
|
|
Term
| How are captured hybrids detected? |
|
Definition
| binding of alkaline phosphatase-labeled anti-DNA/RNA hybrid antibodies in a typical sandwich assay |
|
|
Term
| How is the chemiluminesence measured? |
|
Definition
| by adding the substrate for alkaline phosphate |
|
|
Term
| What is the assay cut off? |
|
Definition
|
|
Term
| what are possible substances of contamination of exogenous alkaline phosphate? |
|
Definition
| detection reagent 1, bacteria, saliva, hair and oils from the skin |
|
|
Term
| Why is it important to clean the work area? |
|
Definition
| nucleic acids are very sensitive to environmental nuclease degradation, nucleases are present on human skin and on the surfaces or materials handled by humans |
|
|
Term
| Can this test differentiate HPV types or infection with more than one type? |
|
Definition
|
|
Term
| where can HPV infect a woman? |
|
Definition
| cervix, vagina, vulva, urethra or area around anus |
|
|
Term
| What makes HPV high or low risk? |
|
Definition
| depends on their known association / lack of association with cancer and its precursor lesion, high-grade cervical intraepithelial neoplasia (CIN 2-3) |
|
|
Term
| how can cervical infections with HR-HPV be detected? |
|
Definition
| Pap screening, colposcopy or biopsy |
|
|
Term
| what is the structure of the HPV virus? |
|
Definition
| Composed of icosohedral viral particle (virion) containing 8000 bp circular dsDNA molecule surrounded by protein capsid |
|
|
Term
| where are the virions found? |
|
Definition
| upper layer only, viral DNA proliferates through entire epithelium |
|
|
Term
| What is the principle of the Hc2 test? |
|
Definition
| Hc2 HR-HPV DNA testing is nucleic acid hybridization assay with signal amplification that utilizes microplate chemiluminescent detection. |
|
|
Term
|
Definition
| hybrid capture- target DNA hybridizes with specific RNA probe, hybrids are captured onto a surface of microplate coated with antibodies specific for RNA-DNA hybrids |
|
|
Term
| What are the steps in specimen denaturation? |
|
Definition
| centrifuge tubes at 3000g for 15 minutes, obtain pellet and decant supernatant, add 150uL of denaturation reagent, vortex to resuspend, place tubes in 65C water bath for 15 minutes, vortex for 30 seconds, run specimen to the 65C water bath for 30 minutes |
|
|
Term
| What are the steps in hybridization? |
|
Definition
| incubate, add probe mix, shake tubes and incubate at 65C for 1 hour |
|
|
Term
| what are the steps in hybrid capture? |
|
Definition
| put sample into pipette, shake, remove liquid by inverting and blot to ensure that all liquid is removed |
|
|
Term
|
Definition
| hybrid detection, immobilized hybrids react with alkaline phosphate conjugated antibodies specific for RNA-DNA hybrids, detected by chemiluminescent substrate, multiple conjugates antibodies bind to each hybrid for signal amplification, as substrate is cleaved off light is emitted by measuring RLUs, intensity off light denotes presence or absence or target DNA in specimen |
|
|
Term
| What are the steps in hybrid detection? |
|
Definition
| add detection reagent 1 into the microplate, cover with lid and incubate |
|
|
Term
| what are the steps in the wash? |
|
Definition
| remove detection reagent from wells, hand wash the wells with buffer, blot plates by inverting, drain and make sure all pink is removed |
|
|
Term
| what are the steps in signal amplification? |
|
Definition
| add detection reagent 2 (yellow), covver and incubate, read the microplate after 15 minutes |
|
|
Term
|
Definition
| analyze, take the RLU measurement and equate it to the CO to indicate a positive or negative |
|
|
Term
| what does RLU measurement < CO mean? |
|
Definition
| absence of HR-HPV DNA sequences or HPV DNA levels below detection limit of the assay |
|
|
Term
| What are the 4 steps in hybrid capture? |
|
Definition
| 1. denature 2. hybridize 3. detect 4. signal amplification |
|
|
Term
|
Definition
| microbiology, cytology, or molecular labs |
|
|
Term
| Which organisms can be detected with Hc2? |
|
Definition
| C. trachomatis, N. gonorrhoeae, CMV, and HPV |
|
|
Term
| What occurs if the viral target DNA is present? |
|
Definition
| DNA is released from the cells and binds to a single-stranded RNA probe creating a DNA/RNA hybrid |
|
|
Term
| What is the RLU based on? |
|
Definition
| readout from the luminometer |
|
|
Term
| UroVysion tells is a specimen is malignant |
|
Definition
| false, just detects a mutation |
|
|
Term
| Cervista HPV test advantage over Hc2 |
|
Definition
| if positive, it tests specifically for types of 16 and 18 |
|
|
Term
| what pathologic entities can be caused by HPV? |
|
Definition
| genital, palmer and plantar warts, CIN and VAIN and bowenoid papulosis |
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