1050 Unit 4 Flashcards
what is flow cytometry?
a powerful tool to identify and enumerate various cell populations
what was flow cytometry first used for?
to perform CD4+ T-cell counts in HIV-infected individuals
what does flow cytometry measures?
multiple properties of cells suspended in a moving fluid medium
what happens as each cell or particle passes single file through a laser light source?
produces characteristic light pattern that is measured by multiple detectors for scattered light (forward and 90 degrees) and fluorescent emissions if the cell is stained with fluorochrome
what is scattered light in a forward direction measure?
measure of cell size
what does a side scatter determine?
a cells internal complexity or granularity
what does a single parameter histogram show?
chosen parameter (x-axis) vs. number of events (y-axis)
what does a dual-parameter dot plot show?
two parameters against each other
what does a gate do?
isolates a particular region of cells for further analysis
what is immunophenotyping?
laboratory technique that uses antibodies to identify cells by their characteristic antigen expression
why is Quantification of an individuals lymphocyte populations essential?
in diagnosing such conditions in lymphomas, immunodeficiency diseases, unexplained infection, or acquired immune disease such as AIDs
How are lymphoid and myeloid cells identified?
using monoclonal antibodies directed against specific surface antigens
what method is used for immunophenotyping of lymphoid and myeloid populations?
flow cytometry
why have clinical laboratories replaced manual immunoassay procedures with automated immunoassay procedures analyzers?
to allow more accurate, precise and sensitive testing of many analytes
what are some factors to consider in determining which analyzer to use?
deciding whether a batch analyzer or a random-access analyzer can best serve testing needs.
what is incorporated in all stages of the laboratory testing, Pre-analytical, analytical, and post-analytical?
automation
what should be done once a new analyzer is purchased?
a thorough validation of all assays to be performed that must be done to ensure quality
what should occur during a proper validation?
determination of accuracy, precision, reportable range, reference range, analytic sensitivity and analytical specificity
what is accuracy?
refers to tests ability to measure what it actually claims to measure
what is precision?
the ability to consistently reproduce the same result on a particular sample
What are pros and cons of a automated analyzer?
CON–> costly
PRO–> reduce turnaround time and reduce error in testing process
what are characteristics of flow cytometry?
– forward scatter of an interrupted beam of light
–side scatter of the interrupted beam of light
–fluorescence emitted from the cells
Forward light scatter is an indicator of a cell’s what ?
size
what is the single most important requirement for samples to be analyzed on a flow cytometer?
cells must be a single-cell suspension
what is the best explanation for a flow cytometers ability to detect several cells surface markers at the same time?
for each marker, a specific fluorochrome-antibody combination is used
what cell surface markers would be present on a population of T helper (Th) cells?
CD3 and CD4
If an analyzer consistently induces a positive test when the analyte in question is not present, this represents a problem with what?
specificity
what are some clinical applications for flow cytometry?
–fetal hemoglobin
–immunophenotyping of lymphocyte subpopulations
–enumeration of stem cells in a peripheral blood mononuclear cell product
the various signals generated by cells intersecting with a flow cytometry laser are captured by what?
photomultiplier tubes
analysis of flow cytometer data of cells can be filtered in many ways by using what method?
“gating” in a dot plot
A newer flow cytometry technology that has the potential to detect 100 analytes from on sample of blood is called what?
cytometric bead array
many flow cytometry laboratories now use the CD45 marker in combination with SSC in differentiating various populations of WBCs to replace what?
FSC + SSC
which cell surface marker is present on cells seen in hairy cell leukemia?
CD103
CD45 is a pan-leukocyte marker expressed on WBCs in varying levels or amounts of expression, based on what?
maturity and lineage of a cell
what best describes single-parameter histogram?
–a chosen parameter is plotted versus the number of events
–chosen by operator
–y-axis = # of events
–x-axis = parameter to be analyzed
–usually extrinsic paraments
–operator can set a marker to isolate the positive event
–computer then calculates % of positive events within designated markers
what are characteristic of PNH?
–PNH could affect RBC and WBC counts
–patients with PNH usually present with symptoms such as hemolytic anemia
–flow cytometry can diagnose PNH by detecting missing anchor proteins
which type of analyzer allows one to measure multiple analytes from numerous samples, loaded at any time?
random access analyzer
operational considerations when selecting automated analyzers for a laboratory include what?
–reagent stability
–test menu
–stat capability
Analyzers use different methods for mixing, including magnetic stirring, rotation paddles, and forceful dispensing. whichever method is used, it is imperative that…
there is no splashing or carryover between samples
what are some benefits of automation?
–greater accuracy
–reduced turnaround time
–savings on reagents
if an analyzer gets different results each time the same sample is tested, what type of problem does this represent?
precision
what is hypersensitivity?
an exaggerated immune response to antigens that are usually not harmful
–results in cell destruction and tissue injury
Gel and Coombs devised a system that does what?
for classifying hypersensitivity reactions into four types based on the immunologic mechanism involved and the nature of the triggering antigen
why are hypersensitvity type I, II, and III considered antibody-mediated?
because they occur within minutes to hours after exposure to antigen, they are referred to as immediate reaction.
what is hypersensitivity type IV?
cell-mediated response involving T lymphocytes. Because the clinical manifestations do not appear until 24 to 72 hours after contact with antigen, type IV response is also referred to as delayed hypersensitivity
describe hypersensitivity type I
–involves production of IgE antibody to an allergen
–sensitization phase
–activation phase
–Degranulation of mast cells and basophils occurs,. with release of performed and newly synthesized chemical mediators that cause an inflammatory response
–Cytokines produced during the response can cause a late-phase response of prolonged inflammation
preformed mediators that are released from mast cells and basophils include what? what is their function?
–histamine
–eosinophil chemotactic factor of anaphylaxis
–neutrophil chemotactic factor
– proteolytic enzymes (tryptase)
preformed mediators do what?
–cause contraction of smooth muscle in the bronchioles, blood vessels, and intestines
–increased capillary permeability
–chemotaxis of eosinophils and neutrophils
–decreased blood coagulability
describe newly synthesized mediators
–prostaglandins, leukotrienes, and PAF
– potentiate the effects of histamine and other preformed mediators
clinical manifestations of type I hypersensitivity include what?
–localized wheal-and-flare skin reactions
–rhinitis (hay fever)
–allergic asthma
–systemic anaphylaxis (life-threatening)
–food allergies
susceptibility to allergies is based on what?
–genetic factors that affect immune response
–environmental influences such as exposure to infectious organisms
what is the treatment for allegies?
–with drugs such as antihistamines, decongestants, bronchodilators and corticosteroids
–monoclonal anti-IgE antibodies such as omalizumab have been used to block binding of IgE to mast cells and basophils in moderate to severe asthma
–allergy immunotherapy (AIT)
when allergen immunotherapy (AIT) used?
–administered to patients for whom drug therapy and environmental control measures are not successful
–goal of AIT is to induce immune tolerance by administering gradually increasing doses of the allergen through time
what is the preferred method of screening for allergies? Describe
–in vivo skin prick test
– where very small amounts of potential allergens are inject under the skin
-positive test produces a wheal-and-flare reaction within 20 minutes
what method is used for testing allergies when the patient can not tolerate a skin test?
–in vitro testing by noncompetitive solid-phase immunoassays for allergen specific IgE can be performed
–in these assays, patient serum is incubated with a solid phase to which a specific allergen has been attached
–Binding is detected with in an enzyme-labeled anti-human IgE antibody and a colorimetric, fluorescent, or chemiluminescent substrate
what can solid-phase immunoassays for total serum IgE used for?
–used to monitor patients undergoing treatment with AIT or monoclonal anti-IgE
–used to detect patients with certain diseased characterized by elevated IgE (rather than allergens) is attached to solid phase
Describe type II hypersensitivity?
–involves production of IgG or IgM antibodies to antigens on the surface of the host cells
–can destroy the cells through complement-mediated cytolysis, opsonization, and phagocytosis or antibody dependent cellular cytotoxicity (ADCC)
–binding of the antibody to the cell surface antigen can result in dysfunction or overstimulation of the cell.
give examples of type II hypersensitivity that involves cell damage
–autoimmune hemolytic anemia
–transfusion reactions
–hemolytic of disease of the fetus and new born (HDFN)
what is myasthenia gravis?
–type II disorder
–antibody blocks binding of a ligand to cell receptors, causing dysfunction of the cells.
–in contrast, antibodies, in Graves disease stimulate cells after binding to their receptors
Describe the direct antiglobulin test (DAT)
–is used to screen for transfusion reactions, autoimmume hemolytic anemia and HDFN
–in this test, washed patient RBCs are combined with anti-human globulin and observed for agglutination, indicating the presence of IgG or complement components on the cells
Describe the indirect antiglobulin test (IAT)
–used in antibody screening and identification and in cross-matching of blood to prevent transfusion reactions
–also used to type patient RBCs for specific blood group antigens
–method detects in vitro binding of antibody to RBCs after addition of anti-human globulin to cause a visible agglutination reaction
what do cold agglutinins antigens do?
–bind to RBC at temperature below 30 C and cause:
—-> blocking of small vessels on exposure to cold
—->red cell agglutination
—->production of hemolytic anemia
explain production of cold agglutinins antigens
–may be from unknown causes or may be associated with certain infections of B cells/plasma cell lymphoproliferative disorders
how can cold agglutinin titer be determined?
–by incubating patient serum with a dilute suspension of human type O RBCs overnight at 4 C and observing for agglutination
Describe Type III hypersensitivity
– involves the formation IgG or IgM antibody that reacts with small antigen-antibody complexes and precipitates out and deposit on tissue
-C’bind vasoldilation and vasoperemeability increase
–macrophages and neutrophils migrate to the affected areas and release lysosomal enzymes, resulting in tissue damage
describe Arthus reaction
-characterized by depositing of antigen-antibody complexes in the blood vessels of the skin
–classic sample of type III reaction
–localized inflammation characterized by redness and edema
–peaks at 3 to 8 hours
describe type IV hypersensitivity
–cell mediated mechanism that involves the activation of Th1 cells to release cytokines
–therefore, macrophages and other immune cells are recruited to the area, where they induce an inflammatory reaction
–cytotoxic T cells may also cause damage to the target cells involved
–hypersensitivity peaks at 48 to 72 hours
Describe contact dermatitis
–low molecular weight compounds contact skin and has haptens sensitize Th1 cells
–example of type IV reaction
–results from exposure to chemicals released by plants (poison oak), metals (nickel), or hair/cosmetic components that act as haptens when bound to self-proteins
Describe hypersensitivity pneumonitis
–allergic disease of lung parenchyma characterized by inflammation of alveoli and interstitial space
–example of type IV reaction
–results mainly from occupational hazard to inhaled antigens. moldy hay, bacterial or fungal spores, pigeon droppings, compost
what is skin testing used for?
–to detect type IV hypersensitivity responses in contact dermatitis and tuberculin (PPD) testing
–also used to test for functional cell-mediated immunity to common antigens in patients suspected of having immunodeficiency diseases.
–positive results appear in 48 to 72 hours and indicate sensitization to the antigens used in test
describe interferon gamma release assays
–IGRAs
–provide an alternative to tuberculin skin testing to detect latent M. tuberculosis infection
–advantages: increased specificity, clearer result interpretation and faster turnaround time to results
describe interferon gamma release assays
–IGRAs
–provide an alternative to tuberculin skin testing to detect latent M. tuberculosis infection
–advantages: increased specificity, clearer result interpretation and faster turnaround time to results
what do all four types of hypersensitivity represent?
–defense mechanisms that stimulate an inflammatory response to cope with and react to an antigen that is seen as foreign.
–in many cases, antigen is not harmful, but the response to it results in tissue damage
What are immune mediators of all four types of hypersensitivity?
type I–> IgE
type II –> IgG or IgM
type III –> IgG or IgM
type IV –> T cells
What are synonyms of all four types of hypersensitivity?
type I –> anaphylactic
type II –> antibody-mediated cytotoxic
type III –> complex-mediated
type IV –> cell-mediated or delayed type
What are the timing’s of all four types of hypersensitivity?
type I –>immediate
type II –> immediate
type III –> immediate
type IV –> delayed
What are the antigens of all four types of hypersensitivity?
type I –> heterologous
type II –> cell surface; autologous or heterologous
type III –> soluble: autologous or heterologous
type IV –> autologous or heterologous
what is a general characteristic of hypersensitivity reactions?
– an exaggerated immune response to an antigen occurs
what is associated with an increase in IgE production?
–activation of Th2 cells
what would cause a positive DAT test?
–presence of IgG on RBCs
–presence of C3b or C3d on RBCs
a transfusion reaction caused by performed antibody
what are characteristics of type I hypersensitivity?
–release of performed mediators from mast cells
–cell-bound antibody bridged by antigen
–an inherited tendency to respond to allergens
