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Immunology > Mod 5 Immunological Principles in the Laboratory > Flashcards

Mod 5 Immunological Principles in the Laboratory Flashcards

1
Q

What immunological principles are used in the laboratory?

A
  1. Commercial production of antibodies.
  2. Immunohematology.
  3. Agglutination testing & serotyping in microbiology.
  4. Immunoassays.
  5. & Other lab tests that use immunological principles.
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2
Q

What does “in vivo” mean?

A

ADJECTIVE
(of a process) performed or taking place in a living organism. The opposite of in vitro.
ADVERB
in a living organism. The opposite of in vitro.

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3
Q

Where normally do antibodies detect antigens?

A

In vivo, i.e. inside your body.

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3
Q

Where normally do antibodies detect antigens?

A

In vivo, i.e. inside your body.

Note: In vivo antibodies are able to detect specific antigens, mostly those present on pathogens, in an effort to remove any potential invaders.

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4
Q

How is Ab/Ag interactions used in the lab?

A

Their characteristic of being specific is used to detect the presence or absence of an antigen.

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5
Q

What does “in vitro” mean?

A

ADJECTIVE
(of a process) performed or taking place in a test tube, culture dish, or elsewhere outside a living organism. The opposite of in vivo. Example: “in vitro fertilization”
ADVERB
in a test tube, culture dish, or elsewhere outside a living organism. The opposite of in vivo.

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6
Q

What are the two different type of antibodies created for use in the lab?

A
  1. Monoclonal antibodies.

2. Polyclonal antibodies.

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7
Q

What are monoclonal antibodies and the result?

A
  1. Monoclonal antibodies are created from one cell line (mono - “one” and clonal - “cell line”).
  2. Result: Ab’s that are highly specific for a single epitope.
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8
Q

What is the technology called that creates monoclonal “high performing” antibodies?

A

Hybridoma

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9
Q

What is the process of creating monoclonal antibodies using hybridoma?

A
  1. Inject mouse with antigen.
  2. Mouse’s adaptive immune response attacks Ag by producing plasma cells that create specific Ab to the Ag.
  3. One cell is selected.
  4. Plasma cell from mouse is fused with a cancerous plasma cell (they have uncontrolled cell division).
  5. Result is uncontrolled cell division of Ab producing cell. This is hybridoma.
  6. Hybridoma is cloned.
  7. Monoclonal antibodies are purified and made for commercial use.
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10
Q

Once a hybridoma is made and cloned what can it do?

A

It can grow and divide indefinitely.

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11
Q

What are some of the applications of monoclonal antibodies (list 5)?

A
  1. Measuring protein or hormones in the clinical lab.
  2. Immuno-histochemical stains.
  3. Therapy for chronic diseases.
  4. Treat cancers.
  5. Identification of cell surface markers, etc.
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12
Q

What is the main principle of monoclonal antibodies?

A

Identification of a specific epitope using antibodies created from one cell line.

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13
Q

What is an example of use given to identify the location and size of a tumor?

A
  1. Inject radioactively labeled monoclonal antibodies into an individual.
  2. Ab’s will circulate to area of specific tumor antigen expression.
  3. Labeled Ab will attach and location and size of tumor can be measured.
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14
Q

What are polyclonal antibodies?

A

Many different plasma cells are producing slightly different antibodies in response to an antigen injected into an animal so they recognize multiple antigenic determinants.

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15
Q

What issues can occur with polyclonal antibodies?

A
  1. Much less specific than monoclonal so they may cross react.
  2. Typically they are heterogeneous antibodies that lack avidity and/or specificity.
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16
Q

How are polyclonal antibodies typically created?

A
  1. They are created by immunizing animals.
  2. Antigen is administered to an animal (rabbit, pigs, sheep, goats, etc.) & their adaptive immune response creates Ab’s.
  3. The antibodies are isolated and purified from the animal’s serum.
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17
Q

What is the advantage/disadvantage of using animals in the method described to create polyclonal antibodies?

A
  1. Advantage: Allows for a much larger quantity of antibody to be produced in a relatively short amount of time compared to hybridoma technology (which can also be very expensive).
  2. Disadvantage: Specificity is
    compromised because many different plasma cells are producing slightly
    different antibodies.
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18
Q

What is a common application for polyclonal antibodies?

A

Often used in histochemical or cytochemical stains.

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19
Q

What are examples of testing in done within transfusion science / immunohematology?

A
  1. Testing blood donations to ensure there are no infections agents.
  2. Determination of blood group system antigens.
  3. Presence or absence of clinically significant antibodies.
  4. Cross-matching to determine compatibility between donor and recipient.
  5. Other specialty testing.
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20
Q

What is needed for accurate and precise testing results in immunohematology?

A

It is imperative that antigens and antibodies be in appropriate concentrations.

This is referred to as an antigen:antibody curve.

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21
Q

What does the reference to “prezone” or “prozone” mean? Result?

A
  1. If concentration of antibodies is too high compared to the concentration of antigens this is referred to as prezone or prozone.
  2. Result: False negative reactions can occur.
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22
Q

What is “postzone”? Result?

A
  1. If antigens are in higher concentrations then antibodies, —> postzone.
  2. Result: False negative reactions can occur too.
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23
Q

What is the optimum concentration of antibodies to antigens called?

A

Zone of equivalence.

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24
Q

What occurs when you have the “zone of equivalence”?

A

Agglutination can occur.

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25
Q

Why are red cells made to ~3-5%?

A

So that the antigens are in proper concentrations for optimal reactions to occur.

Note: False negative reactions could result in misinterpretations and therefore
dire consequences for the patient.

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26
Q

What are the most clinically significant blood group system antigens?

A
  1. ABO blood group.

2. D of the Rhesus blood group.

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27
Q

What is unique about ABO blood group system antibodies in humans?

A

ABO blood group system antibodies are naturally occurring which is quite unique.

This means a person does not need to be exposed to non self RBCs in order to produce these antibodies.

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28
Q

In the ABO blood group system, what does an individual possess antibodies for?

A

They will possess antibodies to the ABO blood group system antigens they lack.

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29
Q

Name the antigens and antibodies for the ABO blood group (or make a table).

A

Type A, A antigen on RBCs, Anti-B in plasma
Type B, B antigen on RBCs, Anti-A
Type AB, A&B antigen on RBCs, No ABO Antibodies
Type O, Neither A or B antigen on RBCs, Anti-A and Anti-B

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30
Q

When someone says their blood group is O+ or A- what does the + or - refer to?

A

The + or - refers to the D antigen.
If they possess the D antigen they are +.
If they do not possess the D antigen they are -.

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31
Q

How are ABO and D groups tested within the laboratory?

A

A source of known antibodies are used. A suspension of patient red cells is done and an anti-serum is added. If there is agglutination then the red cells have the antigen the anti-serum antibodies were against. E.g. If type A blood is combined with Anti-A it will agglutinate. If another anti-serum is used it will not agglutinate.

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32
Q

What is the blood type if the following blood testing results were obtained:
Testing with Anti-A: +
Testing with Anti-B: 0
Testing with Anti-D: 0

A

A negative.

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33
Q

What is the blood type if the following blood testing results were obtained:
Testing with Anti-A: +
Testing with Anti-B: +
Testing with Anti-D: +

A

AB positive

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34
Q

What is “forward typing”?

A

Forward typing is performed to determine what antigens on the red bloods cells.

35
Q

What is reverse typing?

A

Reverse typing determines which ABO antibodies a patient possesses.

Since the patient plasma is the unknown source of antibody, known antigen must be used for testing.

36
Q

What % of suspensions are used when reverse typing?

A

3-5% suspensions of commercial red cells with A or B antigen are used.

37
Q

What is agglutination interpreted as in forward or reverse typing?

A

Agglutination is interpreted as the presence of the antibody (or antigen).

38
Q

How does someone get D antibodies since they are not naturally occurring?

A

To get D antibodies, someone would have been exposed to non-self red cells through

1) previous blood transfusion
2) previous organ transplant
3) blood exchange during/following a pregnancy

39
Q 
What type does a person with typing tests as follows:
Forward typing:
Anti-A: +
Anti-B: 0
Reverse typing:
A cells: 0
B cells: +
D typing:
Anti-D: 0
A

A negative

40
Q 
What type does a person with typing tests as follows:
Forward typing:
Anti-A: 0
Anti-B: +
Reverse typing:
A cells: +
B cells: 0
D typing:
Anti-D: +
A

B positive

41
Q

Why can the Ag:Ab interactions be seen with ABO and D typing?

A

Because they use IgM antibodies and red blood cells.

  1. IgM antibodies are very large pentamers with ten antigen binding sites, and are therefore able to span greater distances between red blood cells.
  2. Reb blood cells are larger cells (relative to microorganisms) which allows for better visualization of agglutination in order to determine presence or absence of antigens.
42
Q

Is it possible to see agglutination of antibodies with microorganisms without an aide?

A

No, when typing microorganisms it is impossible to see visible agglutination without the introduction of another substance such as latex.

43
Q

How does the lancefield group for Streptococcus sp. detect its presence?

A

Lancefield group for Streptococcus sp.:

  1. Uses commercial prepared latex beads with specific antibodies on their surface.
  2. Suspension of Streptococcus is combined with a solution of antibody coated latex beads, the Ab will attach to the carbohydrate Ag of the unknown streptococcus forming cross-linked aggregates and then the test is read for agglutination.
44
Q

How do you interpret a commercial kit with Ab coated latex beads for streptococcus sp. A, B, C, D, F , and G?

A

There are different latex solutions for each type of streptococcus sp. (A, B, C, etc.). The solution the sample agglutinates with indicates the type of Streptococcus Group it is, e.g. (A, B, C, etc.).

E.g. If sample agglutinates w/ solution A it is Streptococcus Group A, Strep pyogenes, the causative agent of strep throat.

45
Q

Name examples of other pathogens that latex agglutination is used to identify.

A

Examples of other pathogens that are identified using latex agglutination include:

a) Cryptococcus neoformans,
b) Clostridium difficile toxins,
c) E. coli, etc.

46
Q

What is serotyping?

A

Using surface antigens to identify microorganisms. A suspension of unknown microorganism is combined with an antiserum to observe agglutination.

47
Q

What is Kauffman and White Classification used for?

A

Kauffman and White Classification is used to classify Salmonella sp. based on the presence or absence of specific surface antigens.

48
Q

What antigens does the Kauffman and White Classification look at?

A

These antigens include:
O (oligosaccharides on pathogen surface), H (flagellar proteins – motile and non-motile strains),
Vi (virulence seen in pathogenic strains), &
K (capsular).

49
Q

What is the salmonella enterica serotype typhimurim reaction?

A

Salmonella enterica serotype Typhimurim reactions are as follows:
1,4,5,12:i:1,2
(numbers represent the presence of antigens identified in the serotype species).

50
Q

What are immunoassays?

A

Immunoassays are laboratory tests that have the ability to qualitatively and quantitatively measure macromolecules through the use of antigen: antibody interactions. In all of the techniques
there is a known (either antigen or antibody) which is labeled in order to detect the reaction in an effort
to identify and measure the unknown (either antibody or antigen).

Often, the unknown macromolecule is a protein that is being measured with the use of a known antibody.

51
Q

What is a type I immunoassay and its mechanism of action?

A

Noncompetitive immunoassay
Mechanism:
1. A reagent of known labeled antibody is added to sample of unknown antigen.
2. Reagent and sample is mixed, reaction is washed, and then the label is measured.
3. The greater the reading of the label the greater the concentration of unknown
antigen in patient sample (linear relationship).

52
Q

What is an example of a Type 1 noncompetitive immunoassay?

A

Example: Labeled anti-THC is added to patient plasma to determine the presence
and quantity of THC.

53
Q

What is a type II immunoassay?

A

Two-site immunoassay

  1. Non-competitive but uses two antibody-antigen binding reactions.
  2. Fixed antibody to reaction well - unknown antigen (from patient) – labeled antibody.
  3. The greater the reading of the label the greater the concentration of unknown antibody or antigen (linear relationship).
54
Q

What is a type III immunoassay?

A

Competitive and homogeneous
immunoassay
1. A reagent of known unlabeled antibody AND labeled antigen is added to unknown patient sample.
2. The unlabeled antigen from the sample competes with the labeled antigen from the reagent.
3. The greater the reading the higher the concentration of unknown antibody or antigen (direct relationship).
4. The label is inactivated by binding.

55
Q

What is Type IV immunoassay?

A

Competitive and heterogeneous
immunoassay

Similar to Type III because there is competition, but free and bound reagent needs to be removed prior to reading because the label is NOT inactivated by binding.

56
Q

What is an example of an immunological technique that can be used at home?

A

Pregnancy test - determines presence of hCG (human chorionic gonadotropic) hormone.

57
Q

How does a pregnancy test work?

A

When the hormone comes into contact with the antibody an antigen: antibody complex can form which results (in some test methods) in a color change or the formation of a visible line.

If hCG is not present then there would be an absence of color change or line, resulting in a negative test.

58
Q

What is ELISA and what are the steps?

A

ELISA is an example of an immunoassay that can be used to determine the presence or absence of antibody or antigen. For example:

  1. a specific antibody can be fixed to wells in a microtitre plate,
  2. patient plasma is added,
  3. microtitre plate is incubated,
  4. microtitre plate is washed to remove unbound antigen,
  5. enzyme labeled antibody is added,
  6. enzyme substrate is added, and
  7. enzyme activity is measured.

This particular reaction would demonstrate a proportional relationship (the greater the concentration of antigen in the sample the higher the enzymatic activity).

59
Q

Where does C-reactive protein get its name from?

A

Its name comes from C-reactive proteins ability to bind to C-polysaccharide in the cell wall of Streptococcus sp.

60
Q

Is C-reactive protein in serum an specific indication of a certain disease?

A

No, It is non-specific; therefore, it is seen in a variety of diseases and infections such as: trauma, myocardial infarction, stress, rheumatoid arthritis, lupus, monitoring effectiveness of anti-inflammatory therapies, etc.

61
Q

How is C-reactive protein measured?

A

Measurement of CRP can be done with nephelometry, which is testing using light
scatter. CRP from the patient’s serum is mixed with a solution of anti-CRP on latex particles.

This complex will cause light scatter. The greater the scatter the higher the concentration of CRP in the patient’s serum.

This high sensitivity testing is done with the use of instrumentation in the clinical laboratory.

62
Q

What is the reference range of CRP for males and females?

A

The normal reference range for CRP is 0.3 – 8.6 mg/L for males and 0.2 – 9.1 mg/L for females.

Each facility will have slightly different reference ranges based on the
instrumentation being used.

63
Q

If CRP is non-specific, then why is it often tested?

A

Often CRP is used to monitor disease progression/regression.

64
Q

What is immunohistochemistry?

A

Immunohistochemistry (IHC) is a technique preformed in the histopathology lab to detect antigens expressed in cells by using specific labeled antibodies.

65
Q

Give an example of an application for immunohistochemistry to detect disease.

A

Cancerous tumors can be identified in tissues by adding labeled antibodies to a slide, allowing the antibody to bind to the
cancer antigens expressed by the cells, and then reading the reaction (this can be with the use of peroxidase or a flurophore).

66
Q

What is flow cytometry?

A

Flow Cytometry is a technology used in the laboratory to identify specific cell types and
numerate cells within complex cell suspensions on a cell-by-cell basis, allowing the analysis of thousands of cells in seconds.

67
Q

What are the main steps in flow cytometry?

A
  1. Labeled antibodies are added to a blood sample
  2. Sample is forced into a “stream” by the analyzer
  3. Cells pass through a laser one by one
  4. Photodetectors capture readings from the labeled antibody attached to the
    cell markers, and perform a count for example.
68
Q

Name an example of a condition that can be diagnosed with flow cytometry.

A

When flow cytometry uses labeled anti-CD4 that will attach to CD4 on helper T cells; this could be used to determine CD4 cell numbers in someone diagnosed with HIV/AIDS to determine the stage of their disease or the effectiveness of treatment with anti-retrovirals.

69
Q

In brief, how does HIV infect cells and specific cells?

A

HIV infects the CD4 cells by recognizing the CD4 marker on its surface, the virus takeovers over protein replication in the host cell, HIV replicated within the cell, the CD4 cell will be destroyed and HIV will continue to infect neighboring cells.

70
Q

What is the prognosis of HIV/AIDS based on?

A

The prognosis of HIV/AIDS is based on the viral load and the number
of CD4 cells present in the patient.

71
Q

Briefly describe the immune system response and the progress of the virus within the immune system.

A
  1. Initially the immune system will attack HIV by mounting an anti-HIV immune response.
  2. The viral load will go down, however, over time the virus will mutate and slowly replicate within CD4 cells.
  3. Eventually, the viral load will increase and CD4 cell numbers will decrease.
  4. When these immune system cells are in such low quantities the result is often patients becoming fatally infected with opportunistic pathogens.
  5. This final stage of HIV infection is referred to as clinical AIDS.
72
Q

What are ways to get exposed to the HIV virus?

A

Exposure to HIV is through blood

contact (IV drug use, unprotected sex, needle stick injury, etc.).

73
Q

How long does it take the average person to generate anti-HIV?

A

It takes two to twelve (2-12) weeks for the average person to generate anti-HIV, so it is important to be aware of the time of exposure to reduce false negative results.

74
Q

What is typically tested for earlier detection of HIV infection?

A

For earlier detection the laboratory can test for HIV antigen between two to
four weeks following exposure.

Many laboratories utilize ELISA testing methods to determine the presence or absence of anti-HIV and/or HIV antigens.

75
Q

Describe the steps involved in the ELISA testing method for anti-HIV or HIV antigens.

A

Testing occurs as follows:

  1. HIV antibody is coated in a reaction well
  2. Patient serum is added
  3. Reaction is incubated
  4. Washed
  5. Enzyme labeled antibody is added
  6. Substrate is added
  7. Enzymatic activity is read
    a) A high concentration of enzyme activity is interpreted as the presence of HIV. Therefore, the patient is infected with HIV.
    b) No activity is interpreted as the absence of HIV.
76
Q

What is hepatitis?

A

Hepatitis is defined as inflammation of the

liver.

77
Q

What are the range of symptoms that someone with hepatitis can experience?

A

In some cases of hepatitis, patients may

exhibit jaundice and in severe cases may result liver failure, while other patients can be asymptomatic.

78
Q

What are some causes of hepatitis?

A

Hepatitis may have several different causes that are viral or non-viral (ex. alcohol and/or drugs).

79
Q

How many hepatitis viral agents implicated in the disease and what are they?

A

There are six different hepatitis viral agents implicated in hepatitis each leading to a different level of severity in the patient:

  1. hepatitis A virus (HAV),
  2. hepatitis B virus (HBV),
  3. hepatitis C virus (HCV),
  4. hepatitis E virus (HEV),
  5. hepatitis G virus (HGV).
80
Q

Which hepatitis virus is of great concern and why?

A

Hepatitis B virus (HBV) is of great concern due to its mode of transmission, exposure to infected body fluids. The virus will quickly increase in numbers and replicate until the immune system recognizes it and can wage war against it.

81
Q

What antibodies are released by the body to fight HBV and ~ when?

A 
IgM antibodies (anti-HBV IgM) will be released approximately two weeks following exposure and will cause viral levels to decrease. This occurs 
in acute viral hepatitis infections. Eventually, the plasma cells will create IgG against HBV (anit-HBV IgG) which will provide long term immunity.
82
Q

How can the knowledge of antibodies and the timing of their deployment by the body help understand the progression of a HBV infection?

A

Determining the class of antibody can help determine whether a patient is fighting a current infection or has long term immunity.

E.g. anti-HBV IgM - current infection, relatively recent.
anti-HBV IgG - longer term immunity

83
Q

What do HBV vaccinations utilize?

A

Vaccinations to HBV utilize HBV surface antigens to stimulate the adaptive immune response and prevent infection. This is a common vaccination required for hospital workers.

84
Q

What does the lab test for when determining if an active HBV infection is present or not prior to the body making Abs?

A

When testing for an active infection prior to the immune system producing antibodies, the lab will test for antigens of HBV (HBV surface antigens- HBsAg) or HBV core antigens (HBcAg).

Immunology (12 decks)

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