Clinical Specimen Basics Flashcards
Multiple diagnostic methods can be used depending on the organism. Describe the methods of direct observation and culture?
Direct observation: stain is quick but lower in sensitivity
Culture is common for bacteria, fungi, and occasional viruses
Parasites are best detected using what diagnostic method?
Detected best by direct observation but immunoassay and molecular assays useful for some
Larger tissue parasites may require biopsy or body imaging
Immunoassays can detect organisms directly or can be designed to do what?
detect antibody response of host to infection
How many negative tests are needed to exclude some organisms?
3 negative test
dont take all 3 specimens in one day
What is precision?
How consistent the test values are
What is accuracy?
How close are the test values to the true value
In terms of negative and positive results what is the best test ?
fewest number of false positives and negatives
What are the formulas for sensitivity, specificity, PPV and NPV
Sensitivity= a/(a+c) Specificity = d/(b+d) PPV= a/(a+b) (percent of all positive test that are true positives is the PPV) NPV = d/(c+d)
What is a predicted value of a test?
is a measure (%) of the times that the value (positive or negative) is the true value
If the population has a disease prevalence of 1% in the general population, there will be how many true positive and how many false positives per 100 random tests that are 98% sensitive ?
1 true positive
2 false positives
this means that 67% of the positives will be false positives
predictive value of a positive is only 33%
What tests are the key method of detection?
cultures
What are traditional methods?
blood agar, extracts from plants and animals and live animal cells of various species
What are alternatives to cultures?
Immunoassays
- detection of specific microbes using antigen capture immunoassay
- detect of antibodies to microbes using specific antibody capture
IgM is a marker of what? IgG is a marker for what?
IgM: acute infection
IgG: can be a marker of recent or old infection
What is the immunodiagnosis steps of an acute infection?
- Draw an acute serum: within the first week or as soon as possible before 14 days
- Draw a convalescent serum 3-6 weeks after the first serum : look for a 4 fold or greater titer increase due to IgG (usually titers go up 1:64 or more after an acute infection)
- Most tests today use ELISA, so the serum titer is given as an optical density.
What are antibody titer tests?
Results are highest dilution of serum still giving a positive Rx
Larger centralized automated labs will provide what in terms of accuracy and cost of tests
Higher accuracy
lower cost tests
In an emergency setting you need STAT tests, what is a trade off for speed in terms of accuracy and costs?
slight decrease in test accuracy and higher costs per tests
What do you do if you miss the acute titer?
Draw a convalescent titer when titer is high. 3-6 weeks after the start of the infection
a very high convalescent titer, and if the clinical symptoms agree, the infection can be presumptively confirmed
Does a single positive antibody allow for a definitive confirmation of acute infections?
nope exceptions are the chronic infections: Lyme Disease HIV infection Hepatitis infection
In diagnosis of chronic infections the acute phase is usually missed. For HIV and Lyme, you do assays to check for presence of a variety of antibody types to increase test specificity. Why do you test for a panel of antibodies to surface and internal proteins?
helps eliminate false positive reactions due to just one or two cross reacting common antigens between closely related microbes
A western blot procedure for HIV and lyme is one way to produce what?
produce a variety of separate antigens from the virus or bacterium of interest so that these antigens can be used to test for distant antibodies from the patient
For diagnosis of hep B you check for what?
check for several antibody markers for viral antigens and human antibodies against the virus
PCR for hep B virus titer
For HIV and Hep C, you can also check for virus antigen or virus DNA or RNA, how do you check this?
Antigen Immunoassay
PCR for nucleic acid
Ab titer in a baby drops or increases each month after birth?
drops
Check antibody titer for agents suspected (CMV, Toxo). What is the timeline for checking babies?
Test at birth (Cord blood serum)
Test baby again after 3 or 4 months
if baby is negative for infection: IgM should not be present and IgG titer from mother will drop at rate of 50% loss every month and will never go back up
If a baby is positive for infection what will be the IgM and IgG?
IgM will be positive
IgG titers eventually go up
Rising and continous IgM is a marker of what?
Acute Infection
IgG is usually what kind of marker?
lifelong
What are some characteristics of third generation immunoassays?
Very convenient to perform
Sensitivity and specificity still just okay typical of enzyme immunoassays
Single Analyze test
What is the test used for influenza?
Binax NOW influenza
one of the easiest to use, read the result after 15 minutes
Fourth generation assays, what are some features?
Simultaneous detection (antigen and antibody)
Kits made with superior reagents (monoclonal antibodies or recombinant antigens)
New improved detection technology
Name the tests in the order of increasing sensitivity
EIA then RIA/FIA then DELFIA then Nucleic acid amplification
what is an example of nucleic acid amplification test
PCR
What is real time PCR?
Simple to perform due to complex chemistry
A colored light producing reaction occurs in a tube
reaction tubes do not have to be handled or opened after putting in the specimen
Explain tests and timelines for chikungunya and dengue diagnosis
Sometimes you need two types of test for dx:
days 2-6 of illness (PCR is best because it detects circulating virus in blood)
days 7-14, ELISA test for IgM in blood
days 6-8, both tests are Ok
What are some new technologies that bypass culture? (high cost)
Mass sequencing replaces cultures (detect multiple pathogens)
Molecular array chips (multiple microbes)
What is a low cost new technology to supplement culture?
MALDI-TOF
For about the effort and time that it takes to do a gram stain of a bacterial colony, it is now possible to identify the colony
Accuracy higher than traditional culture
In regards to technology, it is important to make sure you patients understand what?
tests are FDA approved with a certain % of false positive and negative reactions as a given
In regards to technology, it is important that tests can be set up to be better screening tests or better _________ tests.
confirmatory
it is unusual to have a single test that does it all
What are the guidelines for specimen handling?
Disinfection Quantity and timing Device for collection and transport Transport time Storage time
If a specimen is rejected by the lab it should be saved by the lab at what temp?
4C
What are situations for rejection of specimens that are beyond salvage?
Misidentification of specimen
Culture specimens received in fixative
Dried out specimen on swab
Quantity insufficient for tests ordered
A sample can be rejected due to serious compromise. what are situations in which the specimen has been compromised?
Improper storage temp
Improper transport medium
Improper transport time
Leaky specimen
What organisms require prompt culture if specified by doctor?
Haemophilus ducreyi
Anaerobes in regular transport tubes
Neisseria gonorrhoeae in joint fluid
