Cell Quiz Review - Dr Magranndrmagrann.com/MLT/Immuno PPT Flashcards Unit 2.docx · Web viewWhy must clinical laboratory personnel learn what hazards exist and the basic safety precautions

Immuno PPT Flashcards Unit 2

What contains a wide variety of safety hazards, many capable of producing serious injury or life-threatening disease?

The clinical laboratory

Why must clinical laboratory personnel learn what hazards exist and the basic safety precautions associated with them, and they must apply the basic rules of common sense required for every day safety?

To work safely in this environment

What are unique to the healthcare environment, and others are encountered routinely throughout life?

hazards

In which laboratory does the most significant hazard exists in obtaining and testing patient specimens?

Immunology laboratory

What is the transmission of microorganisms called?

the chain of infection

Why is an understanding of the transmission—the chain of infection—of microorganisms is necessary?

to prevent infection

The chain of infection requires a continuous link between what three elements?

a source, a method of transmission, and a susceptible host.

What is the most likely source of infection is through?

contact with patient specimens

What is the main concern when handling patient specimens?

The exposure to viruses such as the hepatitis viruses and human immunodeficiency virus (HIV)

What are designed to protect health-care workers from exposure to potentially harmful infectious agents?

safety precautions

What is the ultimate goal of biological safety?

to prevent completion of the chain by preventing transmission

What are designed to protect healthcare workers from exposure to potentially harmful infectious agents?

Safety precautions

Preventing the transmission of microorganisms from infected sources to susceptible hosts is critical in?

controlling the spread of infection

What includes handwashing, wearing personal protective equipment (PPE), isolating highly infective or highly susceptible patients, and properly disposing contaminated materials?

Procedures to prevent transmission of microorganisms

Strict adherence to guidelines published by what organizations is essential?

the Centers for Disease Control and Prevention (CDC) and the Occupational Safety and Health Administration (OSHA)

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What is the number one method of infection transmission?

Hand contact

What should always be done at the following times: before patient contact, when gloves are removed, prior to leaving the work area, whenever the hands have been knowingly contaminated, before going to designated break areas, and before and after using bathroom facilities?

Hands should always be washed

What must be washed according to standard guidelines?

Hands

Wet hands with what kind of water? WarmWhat kind of soap is applied to hands for washing?

Antimicrobial

Rub what together to form a lather, create friction, and loosen debris?

Hands

Clean thoroughly between fingers, thumbs, and under fingernails and jewelry up to the wrist for how long?

15 seconds

Rinse hands in what kind of a position? DownwardWhat is used to dry your hands? Paper towelWhy do you turn off faucets with a clean paper towel?

to prevent re-contamination

What is utilized by laboratorians and includes gloves, gowns or laboratory coats, masks, goggles, and face shields?

Personal protective equipment

What are worn to protect the health-care worker’s hands from contamination by patient body substances and to protect the patient from possible microorganisms on the health-care worker’s hands?

Gloves

What is not a substitute for handwashing?

Wearing gloves

When gloves are removed what must be done?

Hands must always be washed

What comes in a variety, including sterile and nonsterile, powdered and un-powdered, and latex and non-latex?

Gloves

What allergy is increasing among healthcare workers?

Latex

Laboratory workers should be alert for symptoms of reactions following contact with what, including irritant contact dermatitis that produces patches of dry,

Latex

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itchy irritation on the hands, delayed hypersensitivity reactions resembling poison ivy that appear 24 to 48 hours following exposure?True immediate hypersensitivity reactions often characterized by facial flushing and respiratory difficulty following contact with what?

Latex

Handwashing immediately after removal of gloves and avoiding powdered gloves may aid in preventing the development of what?

Latex allergy

Replacing latex gloves with what type of gloves provides an acceptable alternative.

nitrile or vinyl

Any signs of a reaction should be reported to a supervisor, because what type of allergy can be life-threatening?

A true latex allergy

Fluid resistant laboratory coats with wrist cuffs are worn to protect the skin and clothing from contamination by patient specimens in which laboratories?

immunology laboratory

What is worn at all times when working with patient specimens?

Laboratory coats

What must be completely buttoned with gloves pulled over the cuffs?

Laboratory coats

What should be changed as soon as possible if they become visibly soiled, and they must be removed when leaving the laboratory?

gloves and laboratory coats

What must be protected from specimen splashes and aerosols?

The mucous membranes of the eyes, nose, and mouth

What can be used to protect the mucous membranes of the eyes, nose, and mouth?

goggles, full-face plastic shields, and Plexiglas countertop shields

Particular care should be taken to avoid what when removing container tops and when transferring and centrifuging specimens?

splashes and aerosols

Never centrifuge specimens in what type of tube?

uncapped

Never centrifuge specimens in what type of centrifuge?

An uncovered centrifuge

When specimens are received in containers with contaminated exteriors, what must be done to the exterior of the

It must be disinfected or, if necessary, a new specimen may be requested.

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container?What was instituted by the CDC in 1985 to protect health-care workers from exposure to bloodborne pathogens, primarily hepatitis B virus (HBV) and HIV?

Universal precautions (UP)

Under universal precautions, all patients are assumed to be what?

possible carriers of bloodborne pathogens

What may occur by skin puncture from a contaminated sharp object or my passive contact through open skin lesions or mucous membranes?

Transmission of bloodborne pathogens

The guideline recommends wearing what when collecting or handling blood and body fluids contaminated with blood?

gloves

The guideline recommends wearing what when there is danger of blood splashing on mucous membranes?

face shields

The guideline recommends disposing of

what in puncture-resistant containers without recapping?

all needles and sharp objects

What is a modification of universal precautions and is not limited to bloodborne pathogens and considers all body fluids and moist body substances to be potentially infectious?

body substance isolation (BSI)

Personnel should wear what at all times when encountering moist body substances?

gloves

What does not recommend handwashing after removing gloves unless visual contamination is present?

BSI guideline

The major features of UP and BSI have now been combined and are called what?

standard precautions

What should be used for the care of all patients and include provisions for the following:•Handwashing•Gloves•Mask, eye protection, face shields•Gowns•Patient care environment•Environmental controls•Linen•Occupational Health and Bloodborne

Standard precautions

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Pathogens•Patient PlacementWhat should be done after touching blood, body fluids, secretions, excretions, and contaminated items, whether or not gloves are worn?

Wash hands

What should be done immediately after gloves are removed, between patient contacts, and when otherwise indicated to avoid transfer of microorganisms to other patients or environments?

Wash hands

It may be necessary to do what between tasks and seizures on the same patient to prevent cross contamination of different body sites?

wash hands

Where what when touching blood, body fluids, secretions, excretions, and contaminated items?

gloves (clean, nonsterile gloves are adequate)

Change what between tasks and procedures on the same patient after contact with material that may contain a high concentration of microorganisms?

gloves

Remove what promptly after use, before touching non-contaminated items and environmental surfaces, and before going to another patient?

gloves

What must be done immediately to avoid transfer of microorganisms to other patients or environments?

Wash hands

What must be worn during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, and excretions to protect mucous membranes of the eyes, nose, and mouth?

mask and eye protection or a face shield

What is worn to protect skin and to prevent soiling of clothing during procedures and patient-care activities that are likely to generate splashes of blood, body fluids, secretions, or excretions?

gown (a clean, nonsterile gown is adequate)

What has to be appropriate for the activity and amount of fluid likely to be encountered?

gown

Remove a soiled gown as promptly as possible, and wash hands to avoid

microorganisms to other patients or environments

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transfer of what?Handle what kind of patient-care equipment soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures, contamination of clothing, and transfer of microorganisms to other patients and environments?

used

Ensure what type of equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately?

reusable

Ensure that what type of items is discarded properly?

single use

Ensure that what has adequate procedures for the routine care, cleaning, and disinfection of environmental services, beds, bed rails, bedside equipment, and other frequently touched services, and ensure that those procedures are being followed?

hospital

Handle, transport, and process used what soiled with blood, bodily fluids, secretions, and excavations in a manner that prevents skin and mucous membrane exposure and contamination of clothing that avoids transfer of microorganisms to other patients and environments?

Linens

Who is responsible for safeguards and regulations?

OSHA (the Occupational Safety and Health Administration)

What is the rules for used needles? Never recap used needlesPlace a patient who contaminates the environment

in a private room.

All biological waste must be placed in appropriate containers labeled with the biohazard symbol except

urine

1:10 dilution of bleach contains 10ml of bleach diluted to 100ml with waterThe national committee for clinical laboratory standards (NCCLS) states for routine cleaning.

1:100 dilution

1:10 dilution of bleach should be stored

stored in a plastic, not a glass, bottle

Specimens picked up by a courier that are to be shipped to an out-of-the-area laboratory

must follow DOT regulations.

DOT and IATA regulation is all diagnostic specimens require triple packaging for

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shippinglabeling of the outer container changed.The terms “clinical specimen” and “diagnostic specimen” have been replaced with

“biological substances, Category B.”

All sharps must be disposed in puncture-resistant, leak-proof containers labeled with the biohazard symbol.

The number-one personal safety rule when handling needles is

to never manually recap one.

all employers to have a written Bloodborne Pathogen Exposure Control Plan

Any accidental exposure to blood through needlestick, mucous membranes, or nonintact skin

must be documented and reported to a supervisor

Meaning of PEP postexposure prophylaxisWhen skin or eye contact occurs , the best first aid is to immediately flush the area

with water for at least 15 minutes and then seek medical attention.

All chemicals and reagents containing hazardous ingredients in a concentration greater than 1 percent

required to have a material safety data sheet (MSDS) on file in the work area

OSHA requires that all facilities that use hazardous chemicals have

a written Chemical Hygiene Plan available to employees

The effects of radiation are dependent on the particular combination of time, distance, length of exposure, and shielding in effect.

radioactive symbol

Disposal of radioactive waste is regulated by

the Nuclear Regulatory Commission (NRC).

serum may be stored 2°C and 8°C for up to 72 hoursthe dilution factor is exactly the same, this is known as

serial dilution

Serial dilutions are often used to obtain a titer, or indication of an antibody’s strength

Definition of Precipitation Combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.

Definition of Agglutination Agglutination is the process by which particulate antigens such as cells aggregate to form larger complexes when a specific antibody is present.

Affinity The initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or determinant site on the corresponding antigen.

Epitope The protein that sticks upward from the cell membrane of a bacteria or other antigen.

Avidity Antibodies are capable of reacting with antigens that

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are structurally similar to the original antigen. This is known as cross-reactivity.

Relation of binding sites and affinity The number of binding sites times the affinity.

The Zone of equivalence Which the number of multivalent sites of antigen and antibody are approximately equal.

Equivalence The part of the precipitation curve that represents an equal concentration of antibodies and antigens.

Lattice hypothesisHypothesis is based on the assumptions that each antibody molecule must have at least two binding sites, and antigen must be multi-valent.

Prozone Antibody excess, the prozone phenomenon occurs.

Postzone Antigen excess, the postzone phenomenon occurs.

False-negative reaction A false-negative reaction may take place in the prozone due to high antibody concentration.

Turbidimetry Turbidimetry is a measure of the turbidity or cloudiness of a solution.

Nephlometry Nephelometry measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension.

The law of mass action? The equilibrium constant is related to strength of the antigen-antibody binding.

Which is more sensitive, nephelometry or turbidometry?

Nephelometry

When no electrical current is used to speed up this process?

It is known as passive immunodiffusion.

Diffusion means Reactants are added to the gel, and antigen–antibody combination occurs.

Radial immunodiffusion (RID) A single diffusion technique where Ab is put into gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel.

The measurement of radial immunodiffusion (end-point)

The square of the ring diameter is then directly proportional to the concentration of the antigen.

End-point method Antigen is allowed to diffuse to completion, and when equivalence is reached, there is no further change in the ring diameter.

Kinetic method Measurements taken before the point of equivalence is reached.

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The measurement of radial immunodiffusion (kinetic method)

The diameter is proportional to the log of the concentration.

Ouchterlony double diffusion both antigen and antibody diffuse independently through a semisolid medium in two dimensions: horizontally and vertically.

When immune complex formed? The density of the lines reflects the amount of immune complex formed.

Line of identity (Serologic identity) Fusion of the lines at their junction to form an arc represents

Line of nonidentity A pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens share no common epitopes

Line of partial identity fusion of two lines with a spurImmunoelectrophoresis Double-diffusion technique that utilizes electric

current to enhance results (speed, Specificity)Rocket immunoelectrophoresis One-dimension electroimmunodiffusion, an

adaptation of radial immunodiffusionThe end result is a precipitin line that is conical in shape, resembling a rocket, hence the

nameThe height of the rocket, measured The height of the rocket, measured from the well to

the apex, is directly in proportion to the amount of antigen in the sample

Rocket immunoelectrophoresis is more rapid than which test?

RID

How soon are results available for Rocket immunoelectrophoresis?

A few hours

Why is it essential to determine the net charge of the molecules at the pH for the test?

Because it determines the direction of migration

A pH is selected so that antibodies in the gel do not move, but what effect does this have on the antigens?

Antigens become negatively charged

Which anode will the antigens migrate towards after a pH is added?

Positive anode

What technique is used to quantitate immunoglobulins?

Rocket immunoelectrophoresis

In rocket immunoelectrophoresis, what is the pH of the buffer?

8.6

What is a double-diffusion technique that incorporates electrophoresis current to enhance results?

Immunoelectrophoresis

What is the typical source of antigens? SerumWhat compounds are separated from serum through electrophoresis?

Main protein fractions

How long is antiserum incubated for after it is placed in a trough?

18-24 hours

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What causes precipitin lines to develop? Antigen-antibody combinationPrecipitin lines are compared in shape, intensity, and location to normal serum in order to detect?

Abnormalitites

Immunoelectrophoresis can be used as a screening tool for?

Serum proteins, including immunoglobulins

Immunofixation electrophoresis is similar to immunoeelectrophoresis except the antiserum is placed?

Directly to the gel surface

Immunoprecipitates form only when? Specific antigen-antibody combination occursWhat becomes trapped in the gel? Immunoprecipitate complexesWhich antibodies are applied to the patient serum in the gel?

Gamma, alpha, mu heavy chains, and kappa or lambda light chains.

Which type of globulinemias exhibit faintly staining bands?

Hypogammaglobulinemias

How do polyclonal hypergammaglobulinemias stain?

Darkly staining bands in the gamma region

Which type of monoclonal bands produce dark and narrow bands?

Waldenstrom’s macroglobulinemia or multiple myeloma

What process is useful in demonstrating antigens present in serum, urine, or spinal fluid in low concentrations?

immunofixation

What is used as a confirmatory test to detect antibodies to HIV-1?

Western Blot

What type of antigens are used in Western Blot?

HIV

How are the components of the HIV antigens separated?

Electrophoresis

What type of paper are the components transferred to?

Nitrocellulose

What is the purpose of transferring the HIV antigen components to nitrocellulose paper?

To preserve the electrophoresis pattern

How does patient serum react with the nitrocellulose?

Through incubation

What steps are required to detect precipitin bands?

Washing and staining

What is detected in the patient sample? AntibodiesWhat error can cause samples to run off the gel or not separate?

Applying the current in the wrong direction

What two errors can hinder proper separation?

Incorrect pH of the buffer and incorrect electrophoresis time

What must be carefully chosen so that lattice formation and precipitation are possible?

The concentration of antigen and antibody.

What will happen if the antibodies or No visible reaction will occur

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antigens are too concentrated?What does the amount of current influence?

The efficiency of separation

What happens if the current is not strong enough?

Separation may be incomplete

What will happen if the current is too strong?

Heat will be generated and may denature the proteins

What is the visible aggregation of particles caused by combination with specific antibody called?

Agglutination

Antibodies that combine with particles are called?

Agglutinins

Antigens must be exposed and able to bind with the antibody because this reaction takes place where?

On the surface of the particle

What are the two steps in agglutination? Sensitization followed by lattice formationIn order to form antibody bridges or lattices, each particle must have?

Multiple antigenic/determiniant sites

What are the different categories of agglutination reactions?

Direct, passive, reverse passive, agglutination inhibition, and coagglutination

In agglutination, what is the first reaction involving antigen-antibody combination through single antigenic determinants on the particle surface?

Sensitization step

The initial reaction in agglutination is rapid and reversible, it follows which law?

Law of mass action

The second step in agglutination is the formation of cross-links that form?

Visible aggregates

Sensitization can be affected by? the nature of the antibody moleculeWhat determines how much an antibody remains attached?

Affinity and avidity

_____ with a potential valence of 10 is over 700 times more efficient in agglutination that is ______ with a valence of 2.

IgM, IgG

What is a key factor in the initial sensitization process?

The nature of antigen-bearing surface

What happens if epitopes are sparse or obscured by other surface molecules?

They are less likely to interact with an antibody

The second stage of agglutination is dependent on __________ and the relative _________of antigen and antibody.

Environmental conditions, concentrations

Lattice formation is governed by what factors?

Ionic strength, pH, and temperature

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Antibodies must be able to bridge the gap between cells by?

One molecule being able to bind to two different cells

Cells, such as erythrocytes or bacterial cells are difficult to bring together into lattice formation because?

They have a slight negative surface charge

In an ionic solution, red blood cells surround themselves with cat ions to form a/an?

Ionic cloud

In an ionic cloud, the ability to link cells together depends in part on?

The nature of the antibody

Antibodies of the IgG class often cannot bridge the distance between particles because?

They are small and have restricted flexibility

Antibodies of the IgG class have restricted flexibility at the hinge region that may prohibit?

Multi-valent binding

Which class of antibodies have a large diameter and are strong agglutinins?

IgM

Visible reactions with IgG require? Enhancement techniquesIn order for the lattice formation to occur what must be controlled?

Surface charge

What is used to decrease the buffer’s ionic strength?

Low-ionic strength saline (LISS)

What helps to neutralize the surface charge and allows red cells to approach each other more closely?

Albumin in concentration of 5-30%

Other techniques that enhance agglutination include increasing the viscosity, using enzymes, centrifuging, and?

altering the temperature of the pH

Viscosity can be increased by adding which agents?

Dextran or polyethylene glycol

How do dextran and polyethylene work? They reduce the water of hydration around the cellsWhat enhances agglutination by cleaving chemical groups on the surface of cells, thereby decreasing hydration?

Enzymes

What is a physical means to increase cell to cell contact and heighten agglutination

Agitation and centrifugation

All of these techniques are used in which department to better detect antigen-antibody reactions, especially in selecting blood for transfusion?

Blood Bank

The secondary or aggregation phase of antigen-antibody reactions is influenced by?

Temperature

_____ agglutinates best at warmer IgG, IgM

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temperatures than ______ antibodies?Naturally occurring antibodies against the ABO blood groups belong to which class?

IgM

Antibody reactions against the ABO blood group are best run at which temperature?

Room temperature

Antibodies to other human blood groups usually belong to which class?

IgG

Antibody reactions to other human blood groups occur best at what temperature?

37○C

The reactions that occur at 37○C are the most important because?

These reactions will occur in the body

Another physiochemical factor that can be manipulated when performing agglutination reactions is?

pH

Most reactions produce optimal antigen-antibody combination at what pH?

6.5-7.5

What type of reactions are easy to carry out, require no complicated equipment, and can be performed as needed in the laboratory without having to batch specimens?

Agglutination

Batching specimens in done when? The test is expensive or complicatedAgglutination test can be used to identify either?

Antigen or antibody

Are agglutination tests qualitative or quantitative?

Qualitative

Semi-quantitative results can be obtained by performing a?

Dilution

Many agglutination variations exist and can be categorized according to?

The type of particle used

What type of agglutination reaction occurs when antigens are found naturally on a particle?

Direct agglutination

One of the best examples of direct agglutination testing involves what kind of antigens?

Known bacterial antigens

What are the bacterial antigens reacted with?

Diluted patient serum with unknown antibodies

Detection of antibodies is primarily used in the diagnosis of?

Disease

What is a rapid screening test to help determine the possibility of typhoid fever?

Widal test

The antigens used in the Widal test Salmonella O and H antigens

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include?What is a significant finding in the Widal test?

A fourfold increase in antibody titer over time

If an agglutination reaction involves red blood cells then it is called?

Hemagglutination

The best example of hemagglutination occurs in?

ABO blood group typing

Antisera of the _____ type can be used to determine the presence or absence of the A and B antigens.

IgM

What type of agglutination reaction is rarely sensitive?

Hemagglutination

A titer that yields semi quantitative results can be performed in test tubes by making serial dilution of?

Antibodies

How is the titer determined in a serial dilution?

It is the reciprocal of the last dilution with a visible reaction

Positive reactions can be graded to indicate the?

Strength of the reaction

Interpretation of a direct agglutination test is done on the basis of what pattern?

Cell sedimentation

If there is a dark red, smooth button at the bottom of the microtiter well, the result is?

Negative

If the cells are spread across the well’s bottom in a jagged pattern, the result is?

Positive

What can be done to see if the cell button will be re-suspended?

The test tube can be centrifuged

If the cell button is re-suspended with no visible clumping then the result is?

Negative

Hemagglutination kits are now available to detect antibodies to which Hepatitis viruses?

Hepatitis A, B, & C.

What type of agglutination employs particles that are coated with antigens not normally found on their surface?

Passive or indirect agglutination

Erythrocytes, latex, gelatin, and silicates are used for what purpose in passive agglutination?

As the particles coating the antigens

What is the advantage of using synthetic beads?

Consistency, uniformity, and stability

What type of particles are inexpensive, relatively stable, and not subject to cross-reactivity with other antibodies?

Latex

A large number of antibody molecules can be bound to the surface of what type

Latex

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of particles, so the number of antigen binding sites is large?What type of agglutination testing has been used to detect rheumatoid factor, anti-nuclear antibody occurring in lupus, and antibodies to group A streptococcus antigens?

Passive agglutination

Passive agglutination can be used to detect antibodies to which viruses?

Cytomegalovirus, rubella, varicella-zoster, and HIV

Passive agglutination kit are designed to detect which class of antibodies?

IgM

Passive agglutination reactions must be carefully controlled and interpreted because they run the risk of nonspecific agglutination caused by the presence of what types of antibodies?

IgM

Commercial tests are usually performed on?

Disposable plastic or cardboard cards or glass slides

If the kits positive and negative controls do not give the expected results then the test is?

Not valid

Passive agglutination tests are used as ___________ to be followed by more extensive testing if the results are positive?

Screening tools

In what type of agglutination testing are antibodies rather than antigens attached to a carrier particle?

Reverse passive agglutination

Reverse passive agglutination is often used to detect?

Microbial antigens

Numerous kits are available today for rapid identification of _____ from such infectious agents as staphylococcus aureus, Neisseria meningitides, streptococcal groups A & B, Haemophilus influenza, rotavirus, vibrio cholera, and leptospirosis.

Antigens

Rapid agglutination test have found the widest application in detecting soluble antigens in which specimens?

Urine, spinal fluid, and serum

The principle is the same for all these tests: latex particles coated with _____ are reacted with a patient sample containing the suspected ______.

Antibody, antigen

How long does it take to identify an organism with fairly high sensitivity and

Few minutes

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specificity?The use of what has greatly cut down on cross-reactivity?

Monoclonal antibodies

Reverse passive agglutination test are often used on what type organisms?

Organisms that are too difficult to grow in the laboratory

Direct testing of specimens for the presence of __________ has still not reached the sensitivity of enzyme immunoassay.

Viral antigens

Latex agglutination tests are extremely useful when a large amount of viral antigen is present in infant infections such as?

Rotavirus or enteric adenovirus

What type of agglutination testing has been used to measure levels of certain therapeutic drugs, hormones, and plasma proteins?

Reverse passive agglutination

What will cause a false positive reaction because it reacts with any IgG antibody?

Rheumatoid factor

What type of reactions are based on competition between particulate and soluble antigens for limited antibody-combining sites?

Agglutination inhibition reactions

In agglutination inhibition reactions what is an indicator of a positive reaction?

A lack of agglutination

Agglutination inhibition reactions involve _____ that are complexed to _____, which is then attached to a carrier particle.

Haptens, proteins

The patient sample is first reacted with a limited amount of reagent antibody that is specific for?

The hapten

The second step involves adding what type of particles containing the same hapten.

Indicator

If the patient sample has no free hapten, the reagent antibody is able to combine with the carrier particles and produce?

Visible agglutination

In agglutination inhibition, what is a negative reaction?

Agglutination

If agglutination occurs, it indicates that the patient did not have sufficient hapten to _____ the secondary reaction.

Inhibit

What can be attached to the particles? Antigen or antibodyThe sensitivity of agglutination inhibition is governed by?

The avidity of the antibody

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Agglutination inhibition can be a highly sensitive assay used to detect small quantities of antigens such as?

Illicit drugs

What are the indicator particles in hemagglutination inhibition reactions?

Red blood cells

What type of testing has been used to detect antibodies to rubella, mups, measles, influenza, parinfluenza, HBV, herpes, RSV, and adenovirus?

Hemagglutination

Red blood cells have what kind of naturally occurring receptors?

Viral receptors

When virus is present, spontaneous agglutination occurs because?

The virus particles link the red blood cells together

The presence of patient ______ will inhibit the agglutination reaction.

Antibody

To perform a hemagglutination inhibition test, patient serum is first?

Incubated with viral preparation

For the second step in a hemagglutination test, what kind of cells are added to the mixture?

Red blood cells

If _____ is present, this will combine with viral particles and prevent _____.

Antibody, agglutination

A lack of agglutination indicates presence of?

Patient antibody

Because there may be a factor in the serum that causes agglutination, or the virus may have lost its ability to agglutinate, what is necessary?

New controls

What is the name given to systems using bacteria as the inert particles to which antibody is attached?

Coagglutination

What is frequently used in coagglutination because it has a protein on its outer surface, called protein A, which naturally absorbs the FC portion of antibody molecules?

Staphyloccus aureus

What face outward and are capable of reacting with specific antigen?

The active sites

Staphylococcus aureus exhibits greater ____ than latex particles and are more _____ to changes in ionic strength

Stability, refractory

These reactions are difficult to read because?

Bacteria are not colored

Coagglutination is highly specific, but it may not be as ______ for detecting small quantities of antigen, as latex

sensitive

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agglutination.Coagglutination has been used to identify?

Steptococci, Neisseria, vibrio and Haemophilus

What is a technique that detects non-agglutinative antibody by means of coupling with a second antibody?

Antihuman globulin test

What is another name for the antihuman globulin test?

Coomb’s test

What is one of the most widely used procedures in blood banking?

Antihuman globulin test

The key component of the test is antibody to human globulin that is made in?

Animals or by means of hybridoma techniques

Antibody to human globulin will react with?

FC portion of the antibody attached to red blood cells

Because the antihuman globulin is able to bridge the distance between cells that IgG cannot, what takes place?

Agglutination

The strength of the reaction is proportional to?

The amount of antibody coating the red blood cells

What are the two types of Coomb’s test? Direct and indirectWhich test is used to demonstrate in vivo attachment of antibody or complement to an individual’s red blood cells?

Direct antiglobulin test

Direct antiglobulin test is an indicator for what diseases?

Autoimmune hemolytic anemia and hemolytic disease of the newborn

Transfusion reaction is tested by which test?

Direct antiglobulin

Why is the test called direct? Because red blood cells are tested directly as they come from the body

A blood sample is obtained from the patient, the red blood cells are washed to remove any antibody that is not attached, and then the cells are tested directly with antibody to?

IgG or complement components

Antihuman globulin is able to bridge the gap between red blood cells and cause visible agglutination if what is present?

IgG or complement

A positive test indicates? An immune reaction is taking place in the individualWhat is a technique that detects nonagglutinating antibody by means of coupling with a second antibody?

Indirect antihuman globin test

Indirect antihuman globulin test can be used to type patient red blood cells for?

Specific blood group antigens

Indirect Coomb’s test is a two-step process. Washed red blood cells and

37○C

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antibodies are allowed to combine at what temperature?The second step of indirect Coomb’s test involves adding______ to see if a visible reaction occurs.

Anti-human globulin

When performing compatibility testing for a blood transfusion, indirect antiglobulin test is often used to check for the presence of?

Clinically significant alloantibody

What type of sample is used to combine with reagent red blood cells of known antigenicity?

Patient serum

All reactions are run at what temperature to detect clinically significant antibodies?

37○C

The cells are washed, anti-human globulin is added and then?

The tubes are centrifuged

What are some possible errors in performing the Coomb’s test?

Failure to wash cells, improper centrifugfation, failure to add test serum or antihuman globulin, or use of expired reagents

Improper concentration of what may alter the results of an indirect Coomb’s test?

Red blood cells

If the red blood cell concentration is too heavy what can occur?

It will mask agglutination

Agglutination can be read without instrumentation but automation can increase the?

Sensitivity

Many of the instrumentation used is based on what principle that as particles combine, light scatter increases, and the absorbance of the solution increases proportionally?

Turbidimetry

What method has been applied to the reading of agglutination reactions, and the term “particle-enhanced immunoassay” is used to describe such reactions.

Nephelometry

What is one type of instrumentation that uses nephelometry?

PACIA

What involves measuring the number of residual non-agglutinate in particles and a specimen?

Particle-counting immunoassay (PACIA)

These particles are counted by means of a __________ in an optical particle counter similar to one that is designed to

Laser beam

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count blood cells.Techniques for the concentration of antigen, incubation time, temperature, diluent, and the method of reading must be?

Standardized

What possible reactions should always be considered?

Cross-reactivity and interfering antibody

What is caused by the presence of antigenic determinants that resemble one another so closely that antibody formed against one will react with the other?

Cross-reactivity

Most cross-reactivity can be avoided through the use of _________ directed against an antigenic determinant that is unique to a particular antigen.

Monoclonal antibody

What are two interfering antibodies that may produce a false-positive result?

Heterophile antibody

Heterophile antibodies are most often a consideration when red blood cells are used as?

The carrier particle

Patients may have an antibody that is capable of reacting with an antigen on the red blood cell other than?

The antigen that is being tested for

Cross-reactions can be controlled by? Preadsorption of test serum on red blood cells or to remove interfering antigens from red blood cells

If rheumatoid arthritis is suspected, sampled can be treated with a chemical to reduce what?

False positive results due to the IgM rheumatoid factor

The use of positive and negative control sera in agglutination testing cannot rule out?

False-positive reactions

Reagents should never be used when? After the expiration dateBefore each use, what should be evaluated and the manufacturer’s instructions be followed accordingly?

Reagents

The sensitivity and specificity may vary for each?

Kit

Rapidity, relative sensitivity, and the fact that if the sample contains a microorganism, it does not need to be viable are all advantages of?

agglutination

What kind of test are simple to perform and require no expensive equipment?

Agglutination tests

Agglutination tests are extremely portable because they are performed on?

Cards, tubes, or microtiter plates

Agglutination tests can test a wide Antigens and antibodies

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variety of?A negative result in an agglutination test does not rule out the presence of the disease or the antigen because?

It is a screening test

A negative results may occur because? The quantity of the antigen or antibody is below the sensitivity

Agglutination tests are important in identifying what rare organisms?

Francisella and Brucella

Labeled immunoassays are designed for what kind of antigens and antibodies?

For those that are small in size or very low in concentration

How is binding detected in antigens or antibodies that are small in size or low in concentration?

Indirectly by using a labeled reactant

What is the substance to be measured called?

Analyte

Bacteria antigens, hormones, drugs, tumor markers, and specific immunoglobulins are examples of what?

Analytes

How many reactants are labeled with a marker?

Only one, either the antigen or the antibody

Fluorescent, radioactive, chemiluminescent, and enzyme labels are all examples of?

Labeled immunoassays

What are the two major formats for all labeled assays?

Competitive and noncompetitive

All the reactants are mixed together simultaneously in which type of immunoassay?

Competitive immunoassay

In competitive immunoassay the amount of bound label is _______ proportional to the concentration of the labeled antigen?

Inversely

A capture antibody is first passively absorbed to a solid phase in which immunoassay?

Noncompetitive immunoassay

In which immunoassay is a second antibody with a labeled added?

Noncompetitive immunoassay

In noncompetitive immunoassays, the amount of label measured is ________ proportional to the amount of patient antigen.

Directly

Radioactivity, enzymes, fluorescent compounds, and chemiluminescent substances are used as?

Labels

It is essential for the antibody used in any immunoassay to have?

A high affinity

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What type of interaction occurs between individual antigen and antibody molecules in competitive binding assays?

random

The higher the affinity of antibody for antigen, the larger the amount of antigen bound to antibody and what can be more accurately measured?

Specific binding

What should the antibody be very specific for when involved in the reaction?

Antigen

What kind of antibodies have been very beneficial in this regard?


What is used to establish a relationship between the labeled analyte measured and any unlabeled analyte that might be present in patient specimens?

Calibrators, or standards

Differing amounts of standards are added to antibody-antigen mixtures to ascertain _____?

Their effect on binding of the labeled reagent

Most instruments then extrapolate this information and create what to determine the concentration of the unknown analyte?

Best-fit curve

In most assays, once the reaction between antigen and antibody has taken place, what step must take place that is a way of separating reacted from unreacted analyte?

Partitioning step

What do most immunoassays currently use for separation, such as polystyrene test tubes, microtiter plates, glass or polystyrene beads, magnetic beads, and cellulose membranes?

Solid-phase vehicle

If a separation step is employed in an assay, what is critical to the accuracy of the results?

Efficiency of the separation

If this is the case, the bound and unbound fractions are usually separated by what means, including decanting, centrifugation, or filtration?

Physical means

What step is this followed by to remove any remaining unbound analyte?

Washing step

What is the last step common to all immunoassays?

Detection of the labeled analyte

This is accomplished by counting Absorbance

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radioactivity in RIA methods, or by the use of enzymes, fluorescence, or chemiluminescence, which typically measure a change in what by spectrophotometry?A negative control, high and low positive controls, and a blank tube (usually phosphate-buffered saline) are typically run as:

Quality-control samples.

True or False.The number, type, and frequency of controls needed vary among instruments and methodologies.

True.

Radioimmunoassay (RIA) was the type of immunoassay developed.

First.

True or False.125I is the most popular radioactive label.

True.

Name other radioactive labels used in RIA.

131I; 125I; and tritiated hydrogen, or 3H

Which radioactive label can be easily be incorporated into protein molecules?

125I

Why can 125I be detected on a gamma counter?

It emits gamma radiation.

What principle is RIA originally based on?

Competitive binding.

In RIA, the 1 being detected competes with a 2 analyte for a limited number of binding sites on a high-affinity 3 .

1. analyte2. radio-labeled3. antibody

Why are all the binding sites on the antibody occupied?

The concentration of the radioactive analyte is in excess.

How may there be decrease the amount of bound radioactive label on the antibody?

If patient antigen is present, some of the binding sites will be filled with unlabeled analyte.

The amount of label in the bound phase is proportional to the amount of patient antigen present.

Indirectly.

What makes RIA extremely sensitive and precise technique?

It can determine trace amounts of analytes that are small in size.

What are disadvantages of working with RIA?

1. The health hazard involved in working with radioactive substances.2. Low-level waste-disposal problems.3. Short shelf life of some reagents.

What is the purpose of enzymes in immunoassays that react with substrates

Enzymes are used as labels

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to produce breakdown products that may be chromogenic, fluorogenic or luminescent?The following are examples of what? Horseradish peroxidase, glucose-6-phosphate dehydrogenase, alkaline phosphatase, and β-D-galactosidase.

Typical enzymes used in labeled immnoassays

Why are alkaline phosphatase and horseradish peroxidase so often used in labeled immunoassays?

They have the highest turnover (conversion of substrate), high sensitivity and are easy to detect

Enzyme assays are classified as either ____ or _____?

Heterogeneous or homogeneous

Enzyme assays are classified as heterogeneous or homogeneous on the basis of what?

Whether a separation step is necessary

What type of enzymatic immunoassay requires a step to physically separate free from bound analyte?

Heterogeneous enzyme immunoassays

What type of enzymatic immunoassay requires no separation step?

Homogeneous enzyme immunoassays

Why do homogenous enzyme immunoassays not require a separation step?

Because enzyme activity diminishes when binding of antibody and antigen occurs

The first enzyme immunoassays (EIAs) were competitive assays based on the principle of what type of assay?

RIA (radioimmunoassay)

Enzyme activity is inversely proportional to the concentration of test substance. What does this mean?

The more patient antigen is bound, the less enzyme-labeled antigen can attach

Currently which type of immunoassays are more common? Competitive or non-competitive enzyme immunoassays?

Non-competitive enzyme immunoassays

Although competitive tests have high specificity, why are non-competitive enzyme immunoassays more common?

Non-competitive enzyme immunoassays offer high sensitivity and specificity, simplicity and low cost.

Non-competitive assays are often referred to as what?

ELISA (indirect enzyme-linked immunosorbent assays)

In labeled immunoassays, either antigen or antibody may be bound to solid phases such as?

Microtiter plates, nitrocellulose membranes, and magnetic latex beads

When antigen is bound to solid phase, what is done with patient serum?

Unknown antibody is added to it and it is incubated

In labeled immunoassays, after a wash step, what is added?

An enzyme-labeled antiglobulin.

In labeled immunoassays, what is the purpose of the enzyme-labeled

It is a 2nd antibody that will react with any patient antibody that is bound to the solid phase

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antiglobulin after the wash step?What is added after a 2nd wash step in labeled immunoassays?

Enzyme substrate

The amount of enzyme label detected is ____ proportional to the amount of antibody in the specimen

Directly

What method remains the preferred screening method for detecting antibody to HIV, Hepatitis A, Hepatitis C, and Epstein-Barr virus

ELISA

Sandwich immunoassays or capture assays have the antibody bound to what?

The solid phase

Antigens captured in labeled immunoassays must have multiple ____.

Epitopes

In labeled immunoassays, after an appropriate incubation period what is added?

Enzyme-labeled antibody

How does the 2nd antibody in the labeled immunoassay complete the “sandwich”?

The 2nd enzyme-labeled antibody recognizes a different epitope than the solid-phase antibody

Enzymatic activity is ______ proportional to the amount of antigen in the test sample

Directly

These types of assays are best suited to antigens that have multiple determinants, such as antibodies, polypeptide hormones, proteins, tumor markers, and microorganisms, especially viruses.

Capture assays

Use of this type of antibodies has made this a very sensitive test system.


Heterogeneous enzyme assays, in general, achieve a sensitivity similar to that of what?

RIA

Possible problems of Labeled Immunoassays include what?

Nonspecific protein binding or the presence of antibodies to various components of the testing system

Sandwich assays are also subject to the hook effect, which is described as…..

An unexpected fall in the amount of measured analyte when an extremely high concentration is present.

This effect typically occurs in antigen excess, where the majority of binding sites are filled. All patient antigen cannot bind in this case.

Hook effect

If this condition is suspected, what can be done and then retested.

Serum dilutions must be made

These are a relatively new type of enzyme immunoassays.

Membrane-based cassette assays

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Membrane-based cassette assays can be described as this?

Typically these are designed as single-use, disposable assays in a plastic cartridge.

The membrane is usually made from this, which is easily able to immobilize proteins and nucleic acids.

Nitrocellulose

Either antigen or antibody can be coupled to the membrane, and the reaction is read by?

Looking for the presence of a colored reaction product.

Some test devices require the separate addition of patient sample, and these components?

Wash reagent, labeled antigen or antibody, and the substrate

This type of rapid assay combines all the previously mentioned steps into one.

Called immunochromatography

The analyte is applied at one end of the strip and migrates toward the distal end, which contains what?

An absorbent pad to maintain a constant capillary flow rate.

As the sample is loaded, it reconstitutes the labeled antigen or antibody, and the two form a complex that does this.

Migrates toward the detection zone.

An antigen or antibody immobilized in the detection zone captures the immune complex and forms this.

A colored line for a positive result.

The excess of this product migrates to the absorbent pad.

Labeled immunoreactant

Test results are most often qualitative or quantitative?

Qualitative

In which antigen–antibody system no separation step is necessary?

homogeneous enzyme immunoassay

What labeled immunoassays are generally less sensitive, rapid, simple to perform, and adapt easily to automation?

homogeneous

In what labeled immunoassays no washing steps are necessary?


What immunoassays are based on the principle of change in enzyme activity as specific antigen–antibody combination occurs?


When free analyte (antigen) competes with enzyme-labeled analyte for a limited number of antibody-binding sites is called:

competitive assay

What happens with active site on the enzyme, when antibody binds to specific determinant sites on the antigen, resulting in a measurable loss of activity?

the active site on the enzyme is blocked

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Enzyme activity is directly in proportion to what?

concentration of patient antigen

Enzyme activity is directly in proportion to what?

hapten present in the test solution

Why sensitivity of homogeneous immunoassays is far less than that achievable by heterogeneous enzyme assays?

because the amplification properties of enzymes are not utilized

Enzyme immunoassays have achieved a sensitivity similar to what assay without attendant health hazards or waste disposal problems?

RIA

In what assays there is no need for expensive instrumentation, and reagents are inexpensive and have a long shelf life?

Enzyme immunoassays

What is disadvantage of enzyme immunoassays?

some specimens may contain natural inhibitors

The size of the enzyme label may be a limiting factor in the design of what assays?

Enzyme immunoassays

Nonspecific protein binding is potential difficulty in what assays?

Enzyme immunoassays

In what immunoassay fluorophores or fluorochromes absorb energy from an incident light source and emit light of a longer wavelength and lower energy as the excited electrons return to the ground state?

fluorescent immunoassay techniques

What two compounds most often used in fluorescent immunoassay?

fluorescent immunoassay techniques

Fluorescein and rhodamine are usually used in what form?

isothiocyanates

Why fluorescein and rhodamine are usually used in the form of isothiocyanates?

because these can be readily coupled with antigen or antibody

Why fluorescein and rhodamine are used together?

Because their absorbance and emission patterns differ

What compound can be used in fluorescent immunoassay?

phycoerythrin


europium (β-naphthyl trifluoroacetone)


lucifer yellow VS

In fluorescent immunoassay fluorescent tags or labels were first used for what

for histochemical localization of antigen in tissues

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purpose?Technique in which fluorescent tags or labels were first used for histochemical localization of antigen in tissues is called:

immunofluorescent assay (IFA)

What techniques are restricted to qualitative observations involving the use of a fluorescence microscope?


In what technique the amount of fluorescence is graded against a dark background?


Immunofluorescent assay (IFA) is used for rapid identification of what?

microorganisms in cell culture or infected tissue


tumor-specific antigens on neoplastic tissue


transplantation antigens

What assay use antibody conjugated with a fluorescent tag and added directly to unknown antigen that is fixed to a microscope slide?

Direct immunofluorescent assay.

What procedure is used in direct immunofluorescent assay?

After incubation and a wash step, the slide is read using a fluorescence microscope.

What is the purpose of direct immunofluorescent assay?

It is used to demonstrate the presence of pathogens in patient samples.

What is the first step in indirect immunofluorescent assays?

The first step is incubation of patient serum (Ab) with known antigen attached to a solid phase.

What is the second step in indirect immunofluorescent assays?

The slide is washed, and then an anti-human immunoglobulin with fluorescent tag is added forming a sandwich which localizes fluorescence.

What is the purpose of indirect immunofluorescent assays?

Such assays are especially useful in antibody identification in patient samples.

What assay is based on the change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody?

Fluorescence polarization immunoassay (FPIA).

What procedure is used in fluorescence polarization immunoassay (FPIA)?

Incident light directed at the specimen is polarized with a lens or prism so the waves are aligned in one plane.

What happens if a molecule is small, rotates quickly enough and is excited by polarized light?

The emitted light is unpolarized.

What happens if the labeled molecule is bound to antibody and unable to tumble as rapidly?

It emits an increased amount of polarized light.

What is reflected by the amount of It reflects the degree of polarized light.

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labeled analyte that is bound?What is the feature of FPIA? Labeled antigens compete with unlabeled antigens in

the patient sample for a limited number of antibody binding sites.

What is the result of FPIA if patient’s sample contains more antigen?

Then less of the fluorescence-labeled antigen is bound and less of the polarization will be detected.

Why FPIA has been used mainly? To determine concentrations of therapeutic drugs and hormones.

The degree of fluorescence polarization is inversely proportional to___________.

Concentration of the analyte.

What is the main problem with fluorescent immunoassays?

Separation of the signal on the labeled from auto fluorescence produced by different organic substance normally present in serum.

What is another difficulty encountered? The nonspecific bending to substances in serum causing quenching or diminishing of the signal and the amount of fluorescence generated.

What is Chemiluminescence? It is another technique employed to follow antigen-antibody combination.

In Chemiluminescence how is the emission of light caused?

It caused by a chemical reaction, typically an oxidation reaction, producing an excited molecule that decays back to its original ground state.

What are some of the most common substances used?

Luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates.

What produced when these substance are oxidized?

It produced of a higher energy state.

What gives when these intermediates spontaneously return to their original state?

It gives off energy in the form of light.

How the light emitted? May exist as a short-lived flash or for a longer period of time.

This type labeling can be used for ________and _____ assays, because labels can be attached to either antigen or antibody.

Heterogeneous and homogenous.

What formats are most often used in heterogeneous assays?

Competitive and sandwich formats.

How it measured therapeutic drugs and steroid hormones?

By using competitive assays.

When we used the sandwich format? For larger analytes, such as protein hormones.What does have chemiluminescent assays comparable to EIA and RIA?

It have an excellent sensitivity and the reagents are stable and relatively nontoxic.

How false results is may be obtained? If there is lack of precision in injection of the hydrogen peroxide.

What some biological materials can cause quenching of the light emission?

Materials in urine or plasma.

What are the powerful new tools being Molecular diagnostic assays

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used to again information to aid diagnosis and monitoring of disease?What is the basis behind the techniques used in molecular diagnostic assays?

On the detection of specific nucleic acid sequences in microorganisms or particular cells.

Which are the molecular techniques used in the clinical lab to identify unique nucleic acid sequences?

Enzymatic cleavage of nucleic acids, gel electrophoresis, enzymatic amplification of target sequences, and hybridization with nucleic acid probes.

Name the two main types of nucleic acids.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

What carries the primary genetic information within chromosomes found in each cell?

DNA: deoxyribonucleic acid

How do genes result into protein production?

Genes are sequences of DNA that encode information for the translation of nucleic acid sequence into amino acid sequence, resulting in protein production.

How long is the entire sequence of the human genome?

More than 3 billion DNA bases long.

About how many genes are known? 30,000 genes.What is DNA packaged in? Duplicate pairs of chromosomes.Humans have pairs of chromosomes, which gives us a total of chromosomes.

2346

Of those 23 pairs, how many are sex chromosomes?

One.

What is ribonucleic acid (RNA)? An intermediate nucleic acid structure that helps convert the genetic information encoded within DNA into proteins.

DNA is the template for making . RNAThe strand is what travels to the ribosomes in the cytoplasm, and is threaded through the ribosome.

RNA

How many nucleic acids on the RNA code for one amino acid?

Every three (3).

Where is the protein formed from the amino acids?

Ribosome.

What is the main sugar present in DNA? Deoxyribose.Ribose is the primary sugar found in: RNA.True or False.DNA exists primarily as a single-stranded molecule.

False.

True or False.Both DNA and RNA are polymers made up of a string of nucleotides.

True

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What are the nucleotides that make up DNA and RNA?

C = Cytosine,T = Thymine,G = Guanine,A = Adenine,U = Uracil

Pair the appropriate nucleotides to together.C = Cytosine,T = Thymine,G = Guanine,A = Adenine,U = Uracil

C to GA to T (in DNA)

How do the nucleotides pair up in RNA? C to GA to U

The two strands are twisted into an: Alpha helix.Why is the beginning of the first DNA strand is called the 5' end and beginning of the second DNA strand is called the 3' end?

They are named that way because of the bond links the 5th phosphate group of sugar to the third hydroxyl group of an adjacent sugar.

True or False.The 5' end of the second strand is attached to the 3' end of the first strand of DNA.

True.

True or False.There is a 5’ end joined to the 3’ end on both sides of the double strand of DNA.

True.

What type of bond holds the two chains together by binding adjacent bases together?

Hydrogen bonds.

How would you describe the DNA molecule?

The DNA molecule makes it ideal for a clinical specimen by losing its conformational structure only under extremes of heat, pH, or the presence of destabilizing agents.

How would you characterize the replication of DNA?--

DNA is characterized as a semi-conservative process because one strand of the molecule acts as a template for creation of a complementary strand.

How do two daughter molecules result? The do two daughter molecules replicate in the laboratory because the hydrogen bonds that hold the two strands together are relatively weak.

How can the double helix separate? The double helix can easily be separated or denatured using heat or an alkaline solution.

What is the process called when heat is used to separate strands?

The process used to separate strands is known as melting.

What happens when DNA strands cool When DNA strands cool to normal physiological

31


to normal physiological conditions? conditions, complementary strands spontaneously rejoin, or anneal.

What type of techniques are used to study nucleic acids?

Molecular diagnostic techniques are utilized to study nucleic acids.

What are the two major processes that involves the genetic sequence encoded within DNA into a function protein?

The two major processes are called transcription and translation.

Define Transcription? Transcription is the generation of a strand of messenger RNA (mRNA) that codes for the gene that is to be expressed as a protein

Define Translation? Translation occurs when mRNA travels from the nucleus into the cytoplasm of the cell to ribosomes to guide protein synthesis.

Describe all forms of RNA? All forms of RNA are single-stranded polymers with an irregular three-dimensional structure of much shorter lengths than DNA.

What is the purpose of the DNA coding strand?

The coding strand serves as the template for the production of mRNA.

Describe all forms of RNA? All forms of RNA are single-stranded strings that are shorter than DNA.

Define Messenger RNA (mRNA)? Messenger RNA (mRNA) is used to translate the DNA code into making functional proteins.

Define Transfer RNA (tRNA)? Transfer RNA (tRNA) transports amino acids to make proteins.

Define Ribosomal RNA (rRNA) Ribosomal RNA (rRNA) acts as the site for protein synthesis directed by the mRNA.

What forms the basis for techniques that are used to detect and characterize genes?

Spontaneous pairing of complementary DNA strands forms these techniques.

What technique is used for identification of individual genes or DNA sequences?

The probe technology is used to identify individual genes and DNA sequences.

Describe a nucleic acid probe? A nucleic acid probe is a short strand of DNA or RNA of a known sequence that is used to identify the presence of its complementary DNA or RNA in a patient specimen.

What is hybridization? Hybridization is binding of two such complementary strands and is very specific.

How many consecutive bases are used to perfect complementarity to form a stable hybrid?

Two strands should share at least 16 to 20 consecutive bases.

What is the probability match occurring by chance?

The probability of such a match occurring by chance is less than 1 in 1 billion.

Four types of probes that are labeled with a marker to make detection of hybridization are?

The four types of probes are radioisotope, fluorochrome, enzyme, and chemiluminescent substances.

What two types of assays does Hybridization can take place in a solid support

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Hybridization take place? medium or in a solution.What two types of nucleic acids utilize in the molecular diagnostic techniques?

Two types of nucleic acids are both DNA and RNA.

What are the simplest types of solid support hybridization assays?

Dot-Blot and sandwich hybridization assays.

What assay provides clinical samples directly to a membrane surface?

The dot-blot assay

How is the membrane denatured on separated by DNA strands?

The membrane is heated to denature or separate DNA strands, and then labeled probes are added.

What two types of assays are used to detect presence of remaining probe?

The two types of assays are enzyme or autoradiography assays.

What does a positive result indicate? The positive result a specific sequence of interest.What does qualitative testing indicate? This type of testing indicates presence or absence of

a particular genetic sequenceWhy are samples handled in multiples? Samples are handled in multiples because it is easier

in this matter.Why is there difficulty with the interpretation of weak positive reactions?

It is difficult with the reactions because there can be background interference.

What is the result after of yielding fragments?

The result is separating from each other on the basis of molecular weight and charge by gel electrophoresis.

Where are digested cellular DNA from a patient blood or tissue sample placed?

The samples are placed in wells in an agarose gel and covered with buffer.

What happens to molecules as they migrate through gel?

Molecules will migrate through gel under an electrical field and separated on the basis of molecular weight. Small fragments migrate faster while larger fragments remain closer to the origin.

There are two ways processing the gel? The gel can be stained with ethidium bromide and viewed under ultraviolet light, or specific nucleic acid sequences can be identified through the use of DNA probes.

Define restriction fragment length polymorphisms (RFLPs)?

Restriction fragment length polymorphisms (RFLPs) are differences in restriction patterns.

What happens in the area of restriction enzymes cleave the DNA?

Variations in nucleotides within the genes change.

What happens during a mutation? During a mutation occurrence, many different sized pieces of DNA are obtained.

What RFLP patterns help identify? RFLP pattern can used to identify specific microorganisms to establish strain relatedness, to detect polymorphisms in major histocompatibility complex (MHC) genes, or to obtain a DNA fingerprint of a particular individual.

What is the purpose of analysis of DNA fragments using probes?

This technique is known as a Southern Hybridization assay, or Southern Blot, named after its discoverer, E.M. Southern.

What kind of solution is used to denature Fragments are denatured using alkali.

33


fragments?What type of membrane are placed after being transferred?

The fragments are transferred onto a nitrocellulose or nylon membrane for the hybridization reaction.

What is used to cross-link the strands onto the membrane?

Heating or UV light

What is a labeled probe? A labeled probe is sequence in complementary to sequence of interest and then added to the membrane for hybridization.

How is the amount of bound probe determined?

After the membrane is washed, the amount of bound probe remaining is determining by detection of the label.

What are the three types of available probes?

The three types of probes are enzyme, chemiluminescent, and acridinium labels.

What is a Southern Blot? A Southern Blot is useful to analyze alterations of large spans of DNA, where amplification by the polymerase chain reaction (PCR) may not be practical.

What do Southern Blot analysis reveal? Southern Blot analysis reveal polymorphisms in DNA sequence based on the RFLP profile made visible by the probes.

What does Southern Blots help determine?

Southern Blots help determine the clonal composition of lymphocyte populations when a large number of cloned cells are present do rearranged genes specific for T cell receptors or immunoglobulins appear in sufficient quantity to produce a detectable band that is different from the normal DNA.

With the help of Southern Blots testing, what three lymphoid malignancies are indicated?

The three lymphoid malignancy are B cell lymphoma, chronic myelogenous leukemia, or hairy cell leukemia.

What helps distinguish between reactive lymphocytes seen in an inflammatory process and true malignancies?

Detection of a malignant clone.

What type of testing is useful in both diagnosis and classification of T-Cell malignancies?

DNA Testing

DNA testing has another impact on diagnosing another lymphoma disease?

DNA testing helps with diagnosing non-Hodgkin’s Lymphoma.

What is Molecular Testing useful for? Molecular testing is useful for Monitor therapy and remission of lymphomas.

Describe the Southern Blot? The Southern Blot are complex, time-consuming to perform, and require a relatively large sample, which can limit its clinical usefulness.

What is a Northern Blot? A Northern Blot is performed in a similar manner to study RNA.

34


How is the Northern Blot technique performed?

The technique is performed by RNA extracted and separated.

What is the reason for using Northern Blot method?

It is often to determine the level of expression of a particular messenger RNA to see if a gene is actually being expressed.

What type of process does the RNA molecule have to go through before a Northern Blot is performed?

The RNA strand have to be denatured to ensure that is unfolded.

What are probes used for during the Northern Blot technique?

The probes are used to identify the presence of specific bands.

A hybridization assays can also be performed of what type technique?

It can be performed in a solution phase.

Compare the setting on how the solution is reacted?

In this type of setting, both the target nucleic acid and the probe are free to interact in a reaction mixture, resulting in increased sensitivity compared to that of solid support hybridization.

How much does the sample require when performing the solution phase?

It requires a smaller amount of sample, and sensitivity is improved when target DNA is extracted and purified.

What type of solution-phase hybridization assays are adaptable to what labels?

The type of labels are automation and chemiluminescent labels.

How many hours can assays be performed?

The assays can be performed in a few hours.

What is an In Situ Hybridization? It represents a third type of hybridization reaction in which the target nucleic acid if found intact cells.

What kind of tissue are used for this In Situ hybridization procedure?

The formalin-fixed and paraffin-embedded tissue sections are used.

How does In Situ hybridization compare to immunohistochemistry?

This technique is the same except that nucleic acids instead of antibodies are used as probes.

How small do probes need to be, to penetrate cells?

Probes need to be small enough, usually 500 bases to reach the nucleic acid.

What is fluorescent in Situ hybridization (FISH)?

This procedure uses a fluorescent tag.

What is Fluorescent In Situ Hybridization used for?

This technique is used to detect a number of malignancies linked to chromosomal abnormalities.

What are Bio Chips, also called microarrays?

They are very small devices used to examine DNA, RNA, and other substances.

What happens to the nucleic acid before its analyzed?

The nucleic acid is amplified and labeled with a fluorescent tag

Where does Hybridization occur? Hybridization occur on the chip’s surface, allowing thousands of hybridization reactions to occur at one time.

How are fluorescent-tagged hybridized samples detected?

The samples are detected with a fluorescent detector.

How does the intensity of the fluorescent Intensity is proportional to the sequence homology at

35


signal maintain a particular location? a particular locus.How are (SNPs) single-nucleotide polymorphisms detected?

They are identified with the set of SNPs that are directly related to or that cause disease.

How can diseases such as leukemias, tumors, and characterizing microbial agents be distinguished?

Detection of point mutations can be used for classifying these diseases.

How is it determined on low sensitive and specific hybridization techniques?

Techniques are determined by conditions of the reaction itself.

How must two nucleic acid strands keep a perfect complementarity to form a stable hybrid?

Nucleic acid strands must share at least 16-20 consecutive bases.

What must be considered with amplification?

Conditions such as stringency, or correct pairing.

How is stringency affected? Stringency is affected by salt concentration temperature, and the concentration of D’s stabilizing agents such as urea.

What is DNA sequencing? DNA sequencing is considered the “gold standard” for many molecular applications.

How do you provide greater accurancy for both DNA strands?

The sequence should be analyzed on both DNA strands.

How are mutations detected? Patient sequences are compared to known reference sequences.

Why is a PCR amplification include most sequence strategies?

It includes a first step to amplify the region of interest to be sequenced.

What is the name of the reaction to sequencing?

The reaction is based on dideoxy chain termination reaction developed by Sanger and collegues in 1977.

What happens when one of the four dideoxynucleotides are incorporated into a growing DNA chain?

Synthesis is halted.

What type of ring does these nucleotides lack the 3’ and 2’ hydroxyl (OH) group?

The pentose ring.

What happens when you try to incorporate dideoxynucleotide to elongate the DNA chain?

It terminates the chain length

What is the most commonly used for DNA sequencing, similar to PCR?

Cycle sequencing

What are the steps that are involved in cycle sequencing?

The steps involve denaturing, annealing of a primer, chain extending, and terminating by varying the temperature of the reaction.

What are newly generated fragments tagged with?

They are tagged with fluorescent dye and separated, based on size, by denaturing gel or capillary electrophoresis.

What are separated fragments spotted by?

They are spotted by fluorescence detectors as the fragments pass through the detector.

What is DNA sequencing most commonly used for?

DNA sequencing is common to detect mutations

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How is testing such as sequencing of HIV an advantage?

Its informative by taking the information and establishing an appropriate therapy and make treatment decisions

Describe Target amplification systems? The systems are in vitro methods for the enzymatic replication of a target molecule to levels at which it can be readily detected.

What are different types of target amplifications?

Polymerase chain reaction(PCR), transcription mediated amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

What is the most commonly employed technique in clinical laboratories?

PCR (Polymerase Chain reaction)

What type of clinical sample is cellular DNA isolated from?

That is usually the patient’s DNA or DNA from infectious organism.

PCR requires four components for reaction to occur?

They are thermostable DNA polymerase, deoxynucleotides of each base, the DNA of interest containing the target sequence and oligonucleotide primers.

Describe oligonucleotide primers? They are short segments of DNA (20-30 nucleotides long), that are complementary to opposite strands that flank the sequence of interest to be amplified and detected.

What are the three steps that consist with the PCR cycle?

The three steps are denaturing, annealing, and extending at different temperatures.

Describe denaturation? The double-stranded DNA is heated to 95C to separate or denature the DNA into single strands.

What happens during the anneal process? Mixture is cooled to 52C, primers are bound to and complementary sequences separate.

What temperature does elongation take place?

72C

Describe elongation process? The heat-stable DNA polymerase binds the 3’ end of each primer and synthesizes a new strand of DNA using the original sample DNA as a template

What happens during an amplification of PCR products?

An exponential process, under optimal conditions, each cycle resulting in a doubling of product.

How many copies in a target sequence can be detected?

Fewer than 100

How are amplicons detected? Amplicons are detected by using various methodsWhat is one method amplicons are detected?

Amplicons are detected by visualization on agarose gels.

Most clinical applications amplicons are detected by what kind of probes?

Amplicons are detected by using labeled nucleic acid probes.

How can RNA be amplified? RNA can be amplified by adding a step prior to the PCR reaction itself.

What process is used to generate copy DNA (cDNA) using the RNA as a

Enzyme reverse transcriptase

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template?What does (RT-PCR) mean? It means Reverse transcriptaseWhat is Real-Time PCR? Real-Time PCR uses fluorescent dyes or probes and

thermal cycles that can take fluorescence readings during each cycle instead of waiting until all the cycles have been completed before analyzing all the amplicons (end-point PCR)

What is one disease that PCR has become a major use for testing?

The one disease that PCR utilizes is AIDS

Who approves assays for quantitation of HIV-1 in human plasma?

FDA-approved assays

What is considered for detecting hepatitis C virus and monitoring therapy?

RT-PCR

What other additional uses are for PCR? By using PCR, it helps identify Mycobacterium tuberculosis and diagnosing early initial infection with (CMV)

What causes false-positive results? Due to extreme sensitivity of PCR, carryover of previously amplified DNA into unamplified samples may cause these results.

What are the strategies to reduce false positives?

The strategies to reduce false positives include automation with closed test systems to control processes, physically separating areas of sample preparation from amplification areas, use of UV light to inactivate DNA within a hood, and use of 10 percent bleach to decontaminate nonporous work surfaces.

What is transcription-based amplification system (TAS)?

It is the first non-PCR nucleic acid amplification method that amplified an RNA target

What does the two-step reaction process involve?

The two-step process involves generation of cDNA from the target RNA followed by reverse transcription of the cDNA template into multiple copies of RNA

What two other non-PCR target amplification methods have been developed that are currently used in clinical assays?

The two methods are nucleic acid sequence-based amplification (NASBA) and transcription mediated amplification (TMA)

What are the advantages that methods NASBA and TMA have?

They are both isothermal reactions that do not require the use of a thermal cycler.

What does Nucleic acid sequence-based amplification (NASBA) use to isothermally amplify an RNA target?

It uses enzymes reverse transcriptase, RNase H, and bacteriophage T7 DNA-dependent RNA polymerase

What is Transcription-mediated amplification (TMA)?

It uses only two enzymes to drive the reaction: an RNA polymerase and a reverse transcriptase.

How many primers are used to target a particular nucleic acid sequence?

Two primers

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Like NASBA, What is also isothermal? TMA, Transcription-based amplification system

What are three other amplification system that are currently available?

The three are strand displacement amplification (SDA), DNA ligase amplification, or the ligase chain reaction (LCR), and branched DNA (bDNA) signal amplification and all present a unique set of advantages and disadvantages

What type of systems subject to several potential problems?

Amplification

What is caused by potential false positive results?

Contaminated nucleic acids

What can also cause false-negative results?

Inhibiting substances in clinical specimens may affect enzymatic amplification methods

What type of procedures can determine if RNA or DNA from an organism?

Amplification procedures

What are the advantages in automated amplification systems?

They result in less potential for contamination and reduce turnaround times and labor costs

What is the purpose of Flow Cytometry? It has been routinely used to identify infection with HIV, and immune-phenotyping cells (to identify their surface antigen expression) has become a major component of the workload in most clinical immunology laboratories.

Define Flow Cytometry? It is an automated system in which single cells in a fluid suspension are analyzed in terms of their intrinsic light-scattering characteristics and are simultaneously evaluated for extrinsic properties (usually specific surface proteins) using fluorescent-labeled antibodies or probes.

How do fluorochromes help flow cytometers?

By using fluorochromes can simultaneously measure multiple cellular properties.

What is a significant advantage of flow cytometry?

The flow rate of cells within the cytometer is so rapid, thousands of cells can be analyzed in seconds, allowing for the accurate detection of very rare cell.

List the major components of a flow cytometer?

The major components include the fluidics, the laser light source, the optics and photodetectors, and a computer for data analysis and management.

Why is it crucial for cells to pass through the laser one at a time?

It is crucial for cells to pass through the laser in one single life, so cellular parameters to be accurately measured in the flow cytometer.

Describe cell suspension process? Cells are processed into suspension, while the cytometer draws up the cell suspension and injects the sample inside a carrier stream of isotonic saline (sheath fluid) to form a laminar form.

How is the sample stream constrained? It is constrained by the carrier stream and is thus hydrodynamically focus so that the cell pass single file through the intersection of the laser light source

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Each cell is interrogated by a light source that typically consists of one or more small air-cooled lasers is a part of what component of flowcytometry instrumentation?

Laser Light Source

What are cells labeled with that absorbs light across a spectrum of wavelengths and emits light of a lower energy across a spectrum of longer wavelengths?

Fluorochrome, or fluorescent molecule

Each fluorochrome has a distinctive spectral pattern of?

Absorption (excitation) and emission

The choice of fluorochromes to be used in an assay depends on?

The light source used for excitation

What do many flow cytometers utilize to increase flexibility in fluorochrome selection?

A second laser

What happens to light when a cell passes through a laser?

Light is scattered in many directions

The amount and type of light scatter (LS) can provide valuable information about a cell’s?

Physical properties

What two angles is light measured by the flow cytometer?

Forward-angle light scatter (FSC) and orthogonal right angle light scatter (or 90 degree angle light scatter (SSC))

Which light scatter angle measures light scatter at less than 90 degrees?

Forward angle light scatter (FSC)

Forward angle light scatter (FSC) is considered an indicator of?

Cell size

90 degree angle light scatter (SSC) is indicative of?

Granularity, or intracellular complexity of the cell

FSC and SSC values can be used to characterize different cell types and are considered?

Intrinsic parameters

Which parameters of cells require the addition of a fluorescent probe for their detection?

Extrinsic parameters

Fluorescent labeled antibodies bound to the cell are interrogated by the laser or lasers of the cytometer concurrently with what measurements of the cell?

FSC and SSC

FSC, SSC, and fluorescent signals generated by the cells’ interaction with the laser are detected by photomultiplier tubes and detectors within what component of flowcytometry?

Optic system

When fluorescent light reaches the photomultiplier tubes, it creates an electrical current that is converted into a?

Voltage Pulse

The digital signals are proportional to what? Intensity of light detectedOnce the intrinsic and extrinsic cellular properties of many cells have been collected , what is the data digitalized by?

Data analysis and management system

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The data set is ready for analysis by who? The operatorTypically, how many “events” are collected for each sample?

10,000-20,000

What is the first level of data representation called?

Single-parameter histogram

A single-parameter histogram plots a chosen parameter on the x-axis versus what on the y-axis?

The number of events

What can be analysed using this type of graph?

Only a single parameter

The operator can then set a marker to differentiate between what kinds of cells for a particular fluorochrome labeled antibody?

Cells that have low levels of fluorescence (negative) from cells that have high levels of fluorescence (positive)

The computer will then calculate the percentage of what events from the total number of events collected?

negative and positive events

What is the next level of representation in which each dot represents an individual cell or even?

Bivariant histogram, or dual-parameter dot plot

How are two parameters, one on each axis, plotted?

Against each other

Using dual-parameter dot plots, what does the operator draw around a population of interest?

A “gate”

What is analysed using dual-parameter dot plots?

Various parameters (extrinsic and intrinsic) of the cells contained within the gated region

What does this allow the operator to screen out?

Debris and isolate subpopulations of cells of interest

The operator chooses which parameters to analyse on both the x- and y- axes and divides the dot plot into what?

Four quadrants

What is the purpose of the four quadrants? Separate the positives from the negatives on each axisWhat can be determined from detailed phenotypic analysis of a cell population?

Lineage and clonality, as well as the degree of differentiation and activation

What kind of samples are commonly used for analysis?

Whole blood, bone marrow, and fluid aspirates

Whole blood should be collected in what kind of tube that has an anticoagulant good for samples processed within 30 hours of collection?

EDTA tubes

Blood should be stored at what temperature prior to processing?

Room temperature (20-25 )℃

How should blood be prepared before being pipetted into staining tubes?

Well mixed

What type of specimens should be rejected? Hemolyzed or clotted specimensSamples with large numbers of red cells require erythrocyte removal to allow for efficient analysis of which cells?

White cells

Historically, density gradient centrifugation with Ficoll-Hypaque was used to generate a

Lymphocytes or blasts

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cell suspension enriched for what types of cell?What results in this method? Selective loss of some cell populationsHow many erythrocyte lysis techniques are available?

Numerous

Tissue specimens are best collected and transported in tissue medium at what temperature if analysis will be delayed?

Either room temperature or 4℃

The specimen is then disaggregated to form a single cell suspension by which method(s)?

Mechanical dissociation or enzymatic digestion.

Which method, also called “teasing”, is preferred using either a scalpel and forceps, a needle and syringe, or wire mesh screen?

Mechanical disaggregation

What is added to the resulting cellular preparation?

Antibodies

What routine application in the clinical laboratory include immunophenotyping of peripheral blood lymphocytes, enumeration of CD34+ stem cells in peripheral blood and bone marrow for use in stem cell transplantation, and immunophenotypic characterization of acute leukemias, non-Hodgkin’s lymphomas, and other lymphoproliferative disorders?

Flow cytometry

Immunophenotyping by flow cytometry has become an important component of initial evaluation and subsequent post-therapeutic monitoring in what types of disease managment?

Leukemia and lymphoma

Flow cytometric findings have been incorporated into which classifications?

Current leukemia and lymphoma classifications

What are the names of these classifications? Revised European-American Lymphoma Classification in 1994, and the proposed World Health Organization (WHO) classifications

In HIV infected patients, what is the highest volume test performed in the flow cytometry laboratory, because it is used in classifying stages of HIV disease and determining treatment options?

Enumeration of peripheral blood CD4+ T cells

What is an additional example of applications of flow cytometry?

The determination of DNA content, or ploidy status, of tumor cells.

Monitoring patients who have been treated for leukemia or lymphoma for which disease has also become another important application of flow cytometry since statistically significant rare cell events can be easily detected using these techniques?

Minimal residual disease

In what case is flow cytometry much more sensitive than the traditional Kleihauer-

Fetal-maternal hemorrhage

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Betke method to detect hemoglobin F positive cells?What methods is flow cytometry also being used for solid organ transplantation?

Human leukocyte antigen (HLA) typing and cross-matching

In addition to the use of flow cytometry, advances in what technology are becoming more evident in clinical immunology labs with the advent of immunoassay analyzers?

Automated technology

Currently there are how many different types of automated immunoassay analyzers that are capable of performing almost all common diagnostic immunoassays?

Over two dozen

In some cases, how does automation compare to manual methods?

It is more accurate and precise, and may be more sensitive

The likelihood of what is decreased when eliminating manual steps, since the potential for error due to fatigue or erroneous sampling is reduced?

Error

Automation can also help streamline test performance to reduce what?

Turnaround time and the cost per test

There are many factors to consider other than the analytic quality of assays that are available when selecting what kind of instrumented?

Automated instrument

What are the two main types of immunoassay analyzers currently on the market?

Batch analyzers and random access analyzers

Which analyzer can examine multiple samples and provide access to the test samples for the formation of subsequent reaction mixtures?

Batch analyzers

However, what is permitted by batch analyzers?

Only one type of analysis at a time

Why might this be considered a drawback in some cases?

Stat samples cannot be loaded randomly, and there cannot be multiple analyses on any one sample

Partially for those reasons, the next generation of analyzers was designed in a modular system that could be configured to measure what from samples?

Multiple analytes from multiple samples

What are these types of analyzers called, in which multiple test samples can be analyzed and multiple testing can be performed on any test sample?

Random access analyzers

Automation can and does occur in all three stages of laboratory testing. What are the three stages?

Preanalytical, analytical, and postanalytical

Introducing sample, adding reagent, mixing reagent and sample, incubating, detecting, calculating, and reporting read out or results are tasks included during which stage?

Analytical

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Which of these tasks may be automated on various immunoassay analyzers?

All or some of the tasks

Reagent used in automated immunoassay analyzers requires consideration of what factors?

Handling, preparing and storing, and dispensing

Some reagents come ready for use, but if not, the analyzer must be able to do what to reagents before they can be used?

Properly dilute

What do most reagents come with that is read by the analyzer to reduce operator error, so if the wrong reagent is loaded into the analyzer by mistake, the analyzer will detect the error and generate an error message?

Bar codes

After reagents have been added to the samples, the next concern is proper mixing to obtain what?

Reliable results

Magnetic stirring, rotation paddles, forceful dispensing, and vigorous lateral shaking are all analyzers use of which method?

Mixing

Whichever method is used, what is imperative to not do between sample wells to prevent erroneous results?

That there be no splashing

Times incubation is then carried out at what temperatures, or analyzers need to have built-in incubators for temperature-controlled incubation?

Ambient temperatures

What does the detection of the final analyte depend on?

The chemistry involved in the immunoassay

In addition to fluorescence or chemiluminescence, both of which require fluorescence detectors, what other method can be used?

Colorimetric absorption spectroscopy

Regardless of the instrumentation considered, what must always be performed?

Proper validation of new instrumentation or methodology

The laboratory must determine how it will meet which standard regulations for verifying the manufacturer’s performance specifications?

Clinical Laboratory Improvement Amendment (CLIA)

As designated by CLIA, what are the required verifications to be determined for a new method?

Accuracy, precision, analytic sensitivity, analytic specificity, interfering substances, reportable range, and reference intervals

What validation method is referred to as the test’s ability to actually measure what it claims to measure?

Accuracy

Parallel testing with an alternative method or technology is a form of which kind of testing?

Accuracy testing

What validation method is referred to as the ability to consistently reproduce the same result on repeated testing of the same

Precision

44


sample?What types of controls should be included in precision testing?

At least one normal and one abnormal control

What validation method is defined as the lowest measurable amount of an analyte?

Analytic sensitivity

What validation method is defined as the assay’s ability to generate a negative result when the analyte is not present?

Analytic specificity

What validation method is defined as the range of values that will generate a positive result for the specimens assayed by the test procedure?

Reportable range

What validation method is defined as the range of values found in healthy individuals who do not have the condition that is detected by the assay?

Reference interval

Choosing an automated immunoassay analyzer is complicated and depends greatly on what?

The main tasks to be performed in a particular laboratory

What type of analyzers may work best if only one type of testing is performed on a large scale?

Batch analyzers

What type of analyzer allows for more flexibility, including rapid processing of stat samples?

Random access analyzers

In either case, what is required on any new instruments before patient results can be reported with confidence?

Extensive validation

Increased turnaround time for testing, remove the possibility of manual errors, and allow for greater sensitivity in determining the presence of low-level analytes is all made possible by what?

Instrumentation

What is defined as a heightened state of immune responsiveness?

Hypersensitivity reactions

What type of reaction is an exaggerated response to a harmless antigen that results in injury to the tissue, disease, or even death?

Hypersensitivity reaction

British immunologists P.G.H. Gell and R.R.A. Coombs devised a classification system for such reactions based on how many different categories?

Four

Cell-bound antibody reacts with antigen to release physiologically active substance is which type of hypersensitivity reactions?

Type I

What is not involved in type I reactions? ComplementFree antibody reacts with antigen Type II

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associated with cell surfaces in which type of hypersensitivity reactions?What plays a major role in producing tissue damage in type II reactions?

Complement

Antibody reacts with soluble antigen to form complexes that precipitate in the tissues in which type of hypersensitivity reaction?

Type III

Complement plays a major role in producing tissue damage in which type of hypersensitivity reactions?

Type III

Which type of hypersensitivity reaction differs from the other three because sensitized T-cells rather than antibody are responsible for the symptoms that develop?

Type IV

What is not involved in type IV hypersensitivity reactions?

Complement

Which hypersensitivity types have previously been referred to as immediate hypersensitivity because symptoms develop within a few minutes to a few hours?

Types I-III

Which type of hypersensitivity has been called delayed hypersensitivity because its manifestations are not seen until 24-48 hours after contact with antigen?

Type IV

The short lag time, usually seconds to minutes, between exposure to antigen and the onset of clinical symptoms is the distinguishing feature of which type of hypersensitivity?

Type I

What is the key reactant present in type I, or immediate, hypersensitivity reactions?

IgE

In type I hypersensitivity, what are the antigens called that trigger the formation of IgE?

Atopic antigens, or allergens

An inherited tendency to respond to naturally occurring inhaled and ingested allergens with continued production of IgE is referred to as what?

Atopy

Patients who exhibit allergic or immediate hypersensitivity reactions produce a large amount of what in response to a small concentration of

IgE

46


antigen?In type I hypersensitivity, actual antibody synthesis is regulated by the action of what?

Cytokines

In type I hypersensitivity, the tendency to respond to specific allergens appears to be linked to inheritance of what kind of genes?

Major histocompatibility complex (MHC) genes

Various HLA class II molecules, especially HLA-DR2, DR4, and DR7, seem to be associated with a high response to what?

Individual allergens

What are HLA-D molecules known to play a role in?

Antigen presentation

Individuals who possess what particular molecules are more likely to respond to certain allergens?

HLA molecules

In type I hypersensitivity, on which chromosome do some individuals who are prone to allergies exhibit certain variations in the gene?

chromosome 11q

Chromosome 11q codes for receptors for which immunoglobulin that is found on several types of cells?

IgE

It is thought that polymorphisms in the beta chain of these receptors are linked to what?

Atopy

These high-affinity receptors, named FC ६-RI receptors, bind the FC region of the epsilon-heavy chain and are found on what types of cells?

Basophils and mast cells

Langerhans and dendritic cells internalize and process allergens from the environment and transport the allergen-MHC class II complex to local lymphoid tissue, where the synthesis of which immunoglobulin occurs?

IgE

Binding of IgE to cell membranes increases the half-life of IgE from 2 or 3 days up to how many days?

At least 10 days

Once bound, what does IgE serve as on mast cells and basophils?

Antigen receptors

Cross-linking of at least two antibody molecules by antigen triggers the release of what from these cells?

Mediators

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Cross-linking of surface-bound IgE on basophils and mast cells by a specific allergen causes changes in the cell membrane that result in the release of what?

Preformed or primary mediators

Histamine, heparin, eosinophil chemotactic factor of anaphylaxis (ECF-A), neutrophil chemotactic factor, and proteases are included in what type of mediators?

Preformed or primary

In addition to immediate release of preformed mediators, mast cells and basophils are triggered to synthesize certain other reactants from the breakdown of what?

Phospholipids in the cell membrane

These products are responsible for what type of allergic reaction that is seen within 6 to 8 hours after exposure to antigen?

Late-phase allergic reaction

Platelet activating factor (PAF), prostaglandin (PG) D2, leukotrienes (LT) B4,C4, D4, and E4, and cytokines are all included in what part of type I hypersensitivity?

Newly formed mediators

In type I hypersensitivity, what is the most severe type of allergic response?

Anaphylaxis

Anaphylaxis is an acute reaction that simultaneously involves what part of the body?

Multiple organs

What type of allergic response may be fatal if not treated promptly?

Anaphylaxis

Symptoms of anaphylaxis depend on what variables?

Route of exposure, dosage, and frequency of exposure

In type I hypersensitivity, what is the most common form of atopy, or allergy?

Rhinitis

Food allergies are an example of what type of hypersensitivity reaction?

Type I immediate hypersensitivity reaction

In what type I immediate hypersensitivity reaction does airflow obstruction occur due to bronchial smooth muscle contraction, mucosal edema, and heavy mucous secretion?

Asthma

Testing for allergies or immediate hypersensitivity can be categorized in what methods?

In vivo or in vitro

Which testing method involves direct In vivo methods

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skin testing, which is the least expensive and most specific type of testing?Which testing method involves measurement of either total IgE or antigen-specific IgE?

In vitro methods

Which testing methods are less sensitive than skin testing but usually are less traumatic to the patient?

In vitro methods

What was the first test developed for the measurement of total IgE?

Competitive radioimmunosorbent test (RIST)

The RIST used radiolabeled IgE to compete with patient IgE for binding sites on a solid phase coated with what?

Anti-IgE

Why has RIST largely been replaced by noncompetitive solid-phase immunassay?

Expense and difficulty of working with radioactivity

What was the original commercial testing method for determining specific IgE known as?

Radioallergosorbent test (RAST)

Principles of the RAST test remain the same, but newer testing involves the use of what rather than radioactivity?

Enzyme or fluorescent labels

What reactants are responsible for type II hypersensitivity, or cytotoxic hypersensitivity?

IgG and IgM

What antigens are responsible for their formation?

Altered self-antigens or heteroantigens

In type II hypersensitivity, antibody coats cellular surfaces and promotes phagocytosis by both opsonization and activation of what?

Complement cascade

Macrophages, neutrophils, and eosinophils have FC receptors that bind to the FC region of antibody on target cells, thus enhancing what process?

Phagocytosis

Natural killer (NK) cells also have FC receptors, and if these link to cellular antigens, what will result?

Cytotoxicity

Once activated, what will complement coat cells with that will facilitate phagocytosis through interaction with specific receptors on phagocytic cells?

C3b

What generate Complement if the cascade goes to completion?

Cell lysis

What are examples of type 2 hypersensitivity reaction?

Transfusion reaction, hemolytic disease of newborn, and autoimmune hemolytic anemia.

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Why some type 2 reactions is involve destruction of tissues?

Because of combination with antibody

What are examples of some type 2 reactions involve destruction of tissue diseases?

Good pasture’s syndrome, Hashimoto’s gravis, and type1 diabetes mellitus.

What detect direct Coombs’ test of type2 hypersensitivity?

In vivo attachment of antibodies in the patient’s own red cells.

What detect indirect Coombs’ test of type2 hypersensitivity?

In vitro attachment of antibodies in the patient’sserum to a panel of reference red cells with knownantigens on their surface.

What are type3 hypersensitivity reactions?

Type 3 hypersensitivity reaction are similar to type2 reactions since IgG or IgM is involved and destruction is complement mediated, in the case of type3 reactions, the antigen is soluble.

What happen when soluble antigen combines with antibody?

Immune complexes are formed that precipitate out of the serum, and normally such complexes are cleared by phagocytic cells.

What happen if the immune system is overwhelmed?

These complexes deposit in the tissues.

What causes complement binds to these complexes in the tissues?

It causes damage to the particular tissue involved.

Where does precipitating complexes occur?

It occurs in mild antigen excess and these are the ones most likely to deposit in the tissue.

Where this deposition does typically occurs?

In the glomerular basement membrane, vascular endothelium, joint linings, and pulmonary alveolar membrane.

What changes long-term deposition? Loss of tissue elements that cannot regenerate and accumulation of scar tissue.

What is the classic example of a localized type 3 reaction?

Arthus reaction.

How causes the inflammatory response? It causes by antigen-antibody combination and subsequent formation of immune.

What causes if complement is fixed? Attracting neutrophils and causing aggregation of platelets.

What releases neutrophil? Neutrophil releases toxic products such as oxygen containing free radicals and proteolytic enzymes.

What is serum sickness? It is a generalized type 3 reaction that is seen in humans

From where results serum sickness? Serum sickness results from passive immunization with animal serum, usually horse or bovine, used to treat such infections as diphtheria, tetanus, and gangrene.

How triggered type 3 hypersensitivity reactions?

They can be triggered by either autologous or heterologous antigens.

Systemic lupus erythematosus (SLE) and rheumatoid arthritis are examples of ___.

Autoimmune disease.

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In SLE, antibodies are directed against constituents such as _________.

DNA and nucleohistones, which are found in most cells of the body.

Immune complex deposition in SLE are involves multiple organs, but in which organ main damage occurs?

In the glomerular basement membrane of the kidney.

In rheumatoid arthritis, an antibody called rheumatoid factor is directed against what?

Against IgG.

How differs type 4 hypersensitivity from the three types of hypersensitivity?

In sensitized T-cells.

Who plays the major role in its manifestations?

Subpopulation of Th1-cells.

Upon subsequent exposure to the antigen, how many hours takes to develop and to reach a peak?

48-72 hours after exposure to antigen.

When Th1 cells are activated, what do they release?

Cytokines, including IL-3, interferon gamma (IFN-y) tumor necrosis factor-beta (TNF-B) and tumor necrosis factor-alpha (TNF-a).

What are Cytotoxic T-cells also recruited?

They bind with antigen-coated target cells to cause tissue destruction.

What is the type of hypersensitivity reaction?

Allergic skin reaction to bacteria, viruses, fungi, and environmental antigens such as poison ivy typify.

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