Routine bench Flashcards
Carriers used in agglutination testing
Erythrocytes, bacterial cells, inert carriers like latex
Factors that can influence the mechanism of agglutination
pH, length of incubation, ionic strength, temperature, stearic hinderance, zeta potential
Hemagglutination
detects antibodies to erythrocyte antigens
Indirect/passive hemagglutination
detects antibodies to antigens other than on RBCs. Chemicals like chromic chloride, tannic acid and glutaraldehyde can be used to cross-link antigens to the cells.
PM shift collects red and lav top tubes and puts them in the refrigerator with the serology RPR specimens. Why are they now unacceptable for cold agglutinin testing?
Storage at 4 Celsius will result in absorption of the cold agglutinins onto the RBCs. They will be absent from the serum and cause a false negative
A physician orders a cold agglutinin titer and states that the patient had electrolytes ordered that morning. When you get the patient’s serum from Chemistry and get a random lav top from hemo for the cells. Your titer shows a strong, irreversible agglutination in tubes 1-11 and a negative result in 12. Why did this occur?
The cells from the lav top were ABO incompatible with the patient. Tube 12 is a negative control with no serum.
Prozone reaction and appearance in cold agglutinin test
Antibody concentration is greater than that of the antigen so the precipitate may be diminished or absent. The first few tubes would show no agglutination until higher dilutions.
Why are 2 different latex reagents used in the cryptococcus assay?
If only the detection latex is positive, it is a true positive. If the control latex is positive, there is a nonspecific interfering factor
How is cryptococcal infection usually acquired?
Inhalation of fungus into lungs. Associated with aged bird droppings and soils contaminated with these droppings.
What other lab methods are used in the diagnosis of cryptococcal infection?
Fungus culture, microscopic examination, IFA, complement fixation, skin test
What does out CALAS test detect?
capsular polysaccharide antigens of Cryptococcus neoformans in serum and CSF
Why and how is complement inactivated in body fluids prior to testing?
Complement is known to interfere with the reactions of certain tests. Inactivation is the process that destroys complement activity.
Serum = add pronase and place in 56 Celsius water bath from 15 minutes then boil for 5 minutes
CSF = boil for 5 minutes
Heterophile antibody
Heterophile antibodies are produced in response to 1 antigen but will react with an unrelated surface antigen present on cells from different mammalian species
Which heterophile antibody is associated with infectious mononucleosis? Is it absorbed by guinea pig kidney cells or by beef erythrocytes? Why is this important?
Paul-Bunnell heterophile antigen occurs in IM and is absorbed by beef erythrocytes. It differentiates is from heterophile antibodies found in conditions like serum sickness and rheumatic diseases absorbed by kidney cells
What principle is our Sure-Vue heterophile antibody test based on?
Davidsohn Differential test
What population is at a greater rick of developing mono?
In developed countries EBV is seen in adolescence and is symptomatic in older children and adults. In developing countries EBV is seen in early childhood and is asymptomatic
What other organisms can cause an IM-like illness?
CMV, toxoplasma, HIV, adenovirus, rubella. A majority of IM come from an unknown etiology
When do IM heterophile antibody levels peak?
2-3 weeks of infection
What immunoglobulin class is the IM heterophile antibody?
IgM
What hematological findings is often present with IM?
Atypical/reactive lymphocytes
How is flocculation different from agglutination?
Flocculation is a specific type of precipitation that occurs over a normal range of antigen concentration. It has a fine precipitate that stays suspended. Agglutination has an insoluble antigen.
What kind of test is the RPR?
Flocculation
5 components of RPR antigen and their function
Cardiolipin = antigenic component, phospholipid from beef heart
Cholesterol = increase solubility
Lecithin = increase sensitivity and standardize the reaction
Choline chloride = eliminates the need to heat inactivate serum, inactivates the complement
Charcoal = visualizing agent, particles entrapped in Ag-Ab lattice, macroscopic reading
Differences between antigen used in RPR and VDRL tests
Complement inactivation = RPR has choline chloride, VDRL doesn’t
Visualization = RPR uses charcoal (macroscopic reading), VDRL doesn’t (microscopic reading)
RPR vs Syphilis IgG test specificity and sensitivity
RPR = nonspecific, detect reagin on antibody like substance present in syphilis and variety of other diseases, non-treponemal
IgG = specific, detects anti-treponemal antibodies, the antigen is treponemal pallidum subspecies pallidum.
Sensitivity is almost equal
What must be done to rotator before rotating cards?
Make sure speed is 100 RPM
Describe some important details concerning the use of the RPR antigen needle
Check delivery of new needle 60 drops/1 mL
Needle should be free of bubble when dispensing drops
Don’t wipe needle
Rinse with CLRW daily
Primary syphilis and RPR, FTA-ABS and CSF-VDRL
pos RPR, pos FTA, NR VDRL (not usually preformed)
Secondary syphilis and RPR, FTA-ABS and CSF-VDRL
pos RPR, pos FTA, NR VDRL (not usually preformed)
Tertiary syphilis and RPR, FTA-ABS and CSF-VDRL
Pos or NR RPR, pos FTA, pos if neurosyphilis VDRL
Treated syphilis and RPR, FTA-ABS and CSF-VDRL
NR RPR, pos FTA, NR VDRL
2 most common methods for direct detection of T. pallidum
Dark field microscopy and fluorescent antibody testing
Why are serologic tests for syphilis performed rather than direct detection of organism by culture?
Cannot be readily isolated or sustained in all culture. Not available in latent or tertiary stages because there is no lesions
reactivity in RPR
Reactivity level of various treponemal tests may differ because of variation in antigen prep. Success of treatment is based on a 4-fold decrease in titer. Same tests used initially should also be used to monitor treatment.
Principle of the immunodiffusion fungal antibody test
Antibody and soluble antigen are placed in separate wells of diffusion medium and allowed to diffuse outward into the medium. Between the wells, a concentration gradient of each of the components is established ranging from antigen excess to antibody excess. Visual line of precipitate forms at point of equivalence
What is the predominant limitation of the fungal procedure?
Cross reaction between fungi
Identity lines
Reported as pos, makes boomerang shape
non-identity lines
Negative except in Aspergillus, makes X shape
Partial identity line
Reported as pos, make Y shape
What does a positive line of identity indicate?
Indicates that patient antibody was made in response to infection with antigen
Who developed the double diffusion 2 dimension system used in fungal serology?
Ouchterlony
What kind of organism is mycoplasma?
It is a member of the Mollicute class. It is free-living (unlike a virus) but does not have a cell wall (unlike a bacteria)
What is the incidence of disease caused by Mycoplasma pneumoniae?
Leading cause of respiratory infections worldwide. Spread from close contact with infected individuals and has an insidious onset. 5-10% progress to pneumonia. Most common in 5-9 year old’s
Why is penicillin not an appropriate therapy?
There is no cell wall and penicillin attacks the cell wall
What is the principle of the mycoplasma IgM test?
Rapid EIA, patient serum is added to wells, 3 drops of conjugate, 3 drops wash buffer and 2 droops substrate. Look for blue color in center after 5 minutes. Test part contains M. pneumoniae antigen and genes as patient test part
Alternatives for diagnosing mycoplasma infection
Ab detection = complement fixation, ELISA, immunofluorescence, hemagglutination
Culture = no gram stain, dark-field microscopy
Ag detection = PCR
What is the importance of an early definitive diagnosis of rotavirus?
Isolation, rehydration therapy, no antibiotics
What other methods are used to diagnose rotavirus?
EIA, membrane EIA, PCR, EM
HIV is the etiologic agent of what?
AIDS
When do HIV patients develop symptoms?
3-6 weeks after exposure
Asymptomatic period of HIV is characterized by
persistent, low level viremia and a gradual depletion of CD4+ T lymphocytes, leading to severe immunodeficiency, multiple opportunistic infections, malignancies and death
What is the recommended testing protocol for HIV?
HIV 1/2 = Ag/Ab combination assay
HIV 1/2 = ag/Ab differentiation assay
Genotyping
CD4+ counts in flow and HIV quantitative RNA levels to monitor treatment
Geenius HIV assay
Confirmation test
Types of samples for HIV test
Serum and EDTA plasma
HIV assay detects
Antibody
What patient populations does the HIV test used for?
Adults and pediatric patients with reactive HIV antibody screening results
What causes the development of the pink/purple lines in a positive HIV test?
Protein-A-colloid gold which binds to captured antibody in sample
How can you tell if you performed the HIV test correctly
Sample migrates through membrane and a pink/purple line develops in control area that has protein A.
How does the operator ensure proper droplet size when adding buffer from the dropper bottle?
Hold bottle vertically and freely drop
How should the operator handle an “indeterminate” result?
Does not exclude the possibility of early seroconversion of the test subject or a cross-reaction with other retroviruses. Should be repeated on new sample in 2-4 weeks
Which volatile organic substances are validated from gas chromatograph testing?
Isopropanol and Methanol
What common household product contains isopropanol?
rubbing alcohol
What are some consequences of isopropanol ingestion?
Increased levels in blood indicate exposure, result in intoxication and CNS depression, can be fatal
What products contain methanol?
Antifreeze, varnish, shellacs, paints, washer fluid, tobacco, adhesives, homemade distilled spirits
What do increased blood levels of methanol indicate?
Intoxication, CNS depression, metabolic acidosis
Why is only CLRW used for preparing standards?
Water with metallic impurities will contaminate or damage the cell
Why must samples have their lids crimped prior to preparing the next sample?
Prevents evaporation during prep
What might happen if the operator turns on the hydrogen without a column connected to the detector fitting inside the oven?
H could diffuse into oven and explode. Don’t turn on H until after operator has verified the column is connected to detector
What value is considered critical in gas chromatography? What do you do?
> 10 mg/dL, must call doctor
What are the specimen requirements for the APT test?
Stool or gastric fluid with visible blood
How to prepare a 0.25 N NaOH solution
Dissolve 10 g of NAOH pellets in 1000 mL of CLRW or dilute 2.5 mL of 10 NaOH to 100 mL of CLRW
How should the APT specimen look after centrifugation
Supernatant should be pink, if not, the test cannot be performed
What does a pink result indicate
Positive for fetal blood
What does a yellow-brown result indicate?
Positive for maternal blood
