0
Q
Name some Universal Precautions taken in the laboratory
A
- All patient specimens are considered to be biohazardous regardless of diagnosis
- Hand washing
- PPE (gloves, gowns, lab coats, goggles)
- Report all accidents
- Dispose of all needles in puncture proof boxes
- No mouth-to-mouth pipetting
1
Q
Name 3 Historical Events in Blood Banking
A
- 1901 –> Landsteiner –> Discovered A, B, and O
- 1970s –> Monoclonal Antibodies
- WWII –> Plastic bags “Fenwal bags”
2
Q
What is the most common method for blood collection?
A
Venipuncture
3
Q
Why are serum separator tube not able to be used in blood banking?
A
Silicone gel interferes with some tests
4
Q
Why are anticoagulated specimens unacceptable to be used in blood banking?
A
The binding of calcium may interfere with some tests
5
Q
What are the purple top tubes used for (EDTA tubes)?
A
Direct antiglobulin test
6
Q
What is the main color tube top used in blood banking? Why?
A
Red top tube and because there is no anticoagulant added
7
Q
Wharton’s Jelly
- Definition
- Where is it found?
A
- Connective tissue substance coating the cord
2. Found when collecting cord blood specimen (umbilical cord)
8
Q
What must you label specimens with?
A
- Patient’s name
- Identification number
- Date
- Time
- Phlebotomist identification
9
Q
At what temperature can specimens be stored? What is the minimum amount of days a specimen is stored?
A
1-6 degrees C
7 days
10
Q
Antigen (definition)
A
- Any substance that binds specifically to an antibody or T cell (TCR)
11
Q
Immunogen or Agglutinogen (Definition)
A
Any substance that stimulates an immune response and reacts with it
12
Q
What is the difference between an antigen and an immunogen?
A
An antigen can react with an immune response but cannot stimulate one whereas an immunogen is the first to illicit an immune response
13
Q
Epitope or antigenic determinant (definition)
A
Discrete chemical site on an antigen that is recognized by an antibody or T cell
14
Q
Hapten (definition)
A
Low molecular weight molecules that do not stimulate an immune response by themselves
15
Q
What is the importance of haptens binding with carriers?
A
This is to increase their size in order to become a complete antigen (cause an immune response)
16
Q
Immunogenicity (definition)
A
An immunogens ability to stimulate an immune response
17
Q
Characteristics of immunogenicity
A
- Antigen structure and position
- Foreigness
- Dosage
- Route
- Metabolic fate
- Host Factors
18
Q
List the types of antigens that have the highest immunogenicity in order from highest to lowest
A
- Proteins
- Carbohydrates
- Lipids
- Nucleic Acids
19
Q
What types of routes can an antigen take into the body? What is the route that yields the highest immunogenicity?
A
- IV – highest
- Oral
- Inhalation
- Intramuscularly
- Subcutaneously
- Intraperitenially
20
Q
Antibodies/Immunoglobulins (definition)
A
Proteins produced by plasma cells to react against epitopes on antigens
21
Q
What are the major classes of antibodies in blood banking?
A
IgM and IgG
22
Q
What antibody type(s) can cross the placenta?
A
IgG
23
Q
What antibody type(s) are present in secretions?
A
IgA
24
What antibody type(s) fix complement?
IgG and IgM
25
IgG
1. Molecular weight?
2. Heavy chain?
3. Structure?
1. 150,000
2. Gamma
3. Monomer
26
IgM
1. Molecular weight?
2. Heavy chain?
3. Structure?
1. 900,000
2. mu
3. pentamer
27
What is the antibody in highest concentration in a serum sample?
IgG
28
What is the antibody in highest concentration in a body wide sample?
IgA
29
Complete Antibodies (definition)
Warm (37C) or cold (4C) saline agglutinations; usually of the IgM class such as anti-A and anti-B and anti-Lewis
30
Complete Antibodies (preferred characteristics)
1. Class
2. Temperature
3. Medium
1. IgM
2. Cold (4C)
3. Saline
31
Incomplete antibodies (definition)
Most are IgG class and at 37C, react and agglutinate only after treated with additional reagents such as albumin, antiglobulin serum, or enzymes
32
Incomplete antibodies (preferred characteristics)
1. Class
2. Temperature
3. Treatment in order for cross linking?
1. IgG
2. Warm (37C)
3. Additional reagents such as albumin, antiglobulin serum, or enzymes
33
Characteristics of a Primary Response
1. Longer lag time
2. Latent period
3. Slower response
4. IgG disappears quicker from system
5. Forms memory cells
34
Characteristics of Secondary (Anamnestic) Response
1. Shorter lag time (1 day or less)
2. High titer of IgG antibodies
3. IgG sustained longer
35
Alloimmunization--Alloantibody
Antibody produced in response to antigen from the same species
36
Autoantibody
Antibodies attacking self cells
37
Natural Antibody
Antibodies produced naturally (there to respond to things occurring in the body naturally)
Ex. ABO system
38
Passive Antibody
Antibodies given through transfusion/placenta (artificially acquired) --> temporary
--> Ex. Hepatitis B immune globulin
39
Antigen - Antibody Reactions
| 1. Types
1. In vivo (body)
| 2. In vitro (test tube)
40
Antigen - Antibody Reactions
| 1. Characteristics
* Specific
* Firm, but reversible
* Surface reaction
* Fairly rapid
* Reactants combine in varying proportions
* Reaction dependent on attraction between molecular forces
* Particulate antigen size
41
Methods to demonstrate (detect) antibody-antigen reactions
1. Agglutination
2. Hemolysis
3. Precipitation
4. Neutralization
5. Antibody labeling - Fluorescence
6. Complement fixation
7. Inhibition, Elution, Absorption
42
Elution
Removing attached antibodies off of red blood cells
43
Absorption
Removing antibody from serum sample using RBC with appropriate antigen
--Autoantibodies
44
Complement
1. Importance
2. Inactivation
1. Cells can be tested for the presence of complement which would indicate if a previous antigen-antibody reaction has occurred
2. 56 degrees C for 30 minutes
45
Dosage
Variable antigen strength based on quantitative differences based on genetics
46
Factors affecting blood group antigen-antibody reactions
* Antigen dosage
* Red cell genotype
* Red cell age
* Storage temperature
* Red cell suspension
* Antibody
* Avidity
* Antibody storage
* Reaction time
* Reaction temperature
* Centrifugation
* pH
* Ionic strength
* Ratio of antigen and antibody
47
Prozone
Antibody excess
48
Zone of equivalence
Optimal concentration of antibody and antigen for best agglutination
49
Postzone
Antigen excess
50
4+
Solid clump of cells
51
3+
Several large clumps
52
2+
Medium-sized clumps with a clear background
53
1+
Small clumps with a turbid background caused by many free cells
54
Weak (+w)
very small clumps with a turbid background
55
Very weak (+vw)
*Manifest only as a rough suspension when observed macroscopically, but agglutinates four or five cells when viewed microscopically
56
Negative (0)
No agglutination
57
H+
Hemolysis -- Positive; supernate is pink to cherry
58
Stages of Agglutination
1. Sensitization
| 2. Agglutination
59
Sensitization (stages of agglutination)
1. Definition
2. Influenced by...
1. Antigen and antibody combine
| 2. Temperature, ionic strength, pH, time, antibody-antigen ratio
60
J chain (Function)
Holds the monomers together to form dimers/pentamers
61
Secretory component (function)
1. Transport IgA to mucosal membrane
| 2. Protects IgA against proteases and pepsinogens
62
Agglutination (stages of agglutination)
1. Definition
2. Influenced by...
1. Total sum of antigen-antibody binding forming a clump
| 2. Number and position of antigen sites, antibody type, and zeta potential (electrostatic repulsion)
63
Zeta Potential
Electrostatic potential of RBCs
* Red blood cells have a negative charged cloud that repels other RBCs
* *Allows for red blood cells to pass by each other
64
Types of mediums used for agglutination tests
1. Saline (IgM)
2. High protein - Bovine albumin or PEG
3. Enzyme systems: trypsin, papain, ficin, bromelin
4. Low ionic strength saline (LISS)
65
Types of antiglobulin reagents
1. Polyspecific
| 2. Monospecific
66
Polyspecific antiglobulin reagent
Usually contains anti-IgG and anti-complement (C3d)
67
Monospecific antiglobulin reagent
Usually contains either anti-C3b or anti-C3d or anti-IgG
68
Antiglobulin Test or Coombs Test
Antihuman sera made from rabbits by injecting with human sera components. Antibody to human globulin (reacts mainly to IgG and/or complement)
69
Direct Antiglobulin Test (DAT)
1. Definition
2. Steps
1. Red blood cell sensitization in vivo (test to see if RBC are coated with antibody)
2. Wash RBC --> Add antiglobulin --> Look for agglutination
70
Direct Antiglobulin Test (DAT) --> Positive in...
* Hemolytic Disease of the Newborn
* Autoimmune Hemolytic Anemia
* Drug Induced
* Chronic Lymphocytic Leukemia
71
Indirect Antiglobulin Test (IDAT)
1. Definition
2. Steps
1. Red blood cell sensitization in vitro (test patient serum for the presence of antibody by adding reagent RBC and antiglobulin)
2. Incubate patient serum with RBC reagent antigen --> wash --> add antiglobulin --> check for agglutination
72
Indirect Antiglobulin Test (IDAT) --> Positive in...
* Alloimmunization (serum from previous transfusion or pregnancy)
* Acquired hemolytic anemia (drugs)
* Crossmatch, antibody screen
* Detection of antigens
73
Coombs check cells or Control cells
1. Definition
2. Confirmation of...
1. O positive cells coated with anti-D (should see agglutination)
2. Negative DAT or IDAT
74
Transferases
Enzymes that assemble sugars and other complex molecules --> move from source to receptor)
75
Alleles
Alternate genes on a given loci on a chromosome
76
Homozygous
Identical alleles on a given locus
77
Heterozygous
Alternate forms of alleles on a given locus
78
Co-dominant
Expression of both alleles
79
Amorph
Silent gene --> No detectable products
80
H gene
Precursor substrate chain for A and B gene specified transferases to build on
81
H Genes immunodominant sugar
L-Fucose
82
A Gene Immundominant sugar
N-acetyl-D-galactosamine
83
B Gene Immunodominant sugar
D-galactose
84
O gene immunodominant sugar
None
85
Most H antigen (reacts strongly with anti-H) --> Least H antigen (reacts less with anti-H)
O --> A2 --> A2B --> B --> A1 --> A1B
86
Anti-H
Antisera from lectin or seed extract from Ulex europaeus
87
Bombay
1. Definition
2. Characteristics
1. hh or Oh or ABHnull
2. Appear type O since lack H substance but genetically can still be A or B
* *Has anti-H, anti-B, or anti-A
* *No agglutination with anti-H
* *Only Bombay blood is compatible
88
What type of antibodies are anti-A and anti-B?
Natural antibodies (natural immunization)
89
How are anti-A and anti-B acquired?
Response to exposure to similar A and B substances in bacteria and plants (in gut)
90
When are anti-A and anti-B present?
Not usually present at birth; develops in first 2-3 months in life
91
What class of antibodies are anti-A and anti-B?
IgM
92
Forward Type/Front type/Cell type
Patient RBC (antigen) with reagent antibody
93
Reverse type/Back type/Serum type
Patient serum (antibody) with reagent cells
94
Types of blood reagents used in blood banking:
* RBCs with known antigens
* Antisera of known antibody specificity
* Antiglobulin which detects IgG and/or complement
* Potentiators which enhance reaction
* Fresh serum-Fresh complement and increased antibody titers
* Blood samples-No hemolysis before testing
* Lectins-plant proteins that binds specific carbs
95
Importance of fresh serum reagent?
Fresh complement and increased antibody titers
96
Subgroups of A
| 1. Types
1. A1 and A2
97
What lectin is used to determine A1?
Seed extract of Dolichos biflorus
98
What will lectin with anti-A1 react with?
A1 and A1B cells but not A2 or A2B
99
Secretor Gene function
Converts A, B, and H to water soluble so it may be found in the body fluids
100
Secretor Status
Gives a soluble A, B, H substance in body fluids like saliva
101
ABO Discrepancies
1. Unexpected antigen or antibody
2. Lack of expected antigen or antibody
3. Weakened expression of antigen or antibody
102
ABO problems that can occur in the serum
* anti-A1
* auto-antibody
* anti-H
* chimera
* old age
* newborn
* alloantibody
* anti-I
103
ABO problems in cells
* A or B subgroups
* Acquired A or B
* Disease (leukemia)
* Wharton's Jelly
* Chimera
* Polyagglutination
* Dyes
* Contaminants
* Rouleaux
* Panagglutination
104
Methods to resolve an ABO problem
* Repeat ABO type
* Allow backtype to sit for 30 minutes
* Repeat wash of patient cells
* Exam for Rouleaux
* Run autocontrol to detect autoantibody
* Check for anti-A1
* New patient specimen
* Test with anti-A1B, anti-A1, and anti-H
* DAT
* Serum with A1, A2, B, and O cells
* Cell and serums at room temperature and 4C
105
Additional ABO problems:
1. Reverse Type is weak or absent
2. Acquired B antigen
3. O cells given to A or B person
4. A2 with Anti-A1
5. Decreased transferase levels
6. Blood group antigen altered by bacterial enzymes
7. Decreased IgM levels with age, drugs, or disease
8. Unexpected alloaggutinins (anti-M, anti-P, anti-A, anti-Lewis)
106
Reverse Type is weak or absent
1. Causes
2. Procedure to determine
1. agammaglobinemia or hypogammaglobinemia; newborns or elderly
2. Check subgroups of antigen; check if other antibody levels are normal; incubate at 4C; check ABH secretor status
107
Acquired B antigen
1. Associated with...
2. Characteristics
3. How to detect..
1. Colon carcinoma, gram negative infections, intestinal obstructions
2. Seen when type AB with anti-B; anti-A stronger reaction than with anti-B
3. Run autocontrol; test saliva
108
O cells given to A or B person
1. Characteristics
2. Associated with...
1. Mixed field agglutination
| 2. Leukemia, fetal maternal bleed, chimerism
109
A2 with Anti-A1
| 1. Tests
* Test with A, B serum and at 4C
* Check for mixed field agglutination
* Elution and Absorption studies
* Secretor status
110
Chimera
1. Definition
2. Causes
1. Two different populations of red blood cells
| 2. Transfusions and fraternal twins
Blood Banking (BIOL 380)
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