Test 1: Transfusion Practices Flashcards
What type of immunity is Mediated by macrophages, T cells, and
dendritic cells?
Innate or Natural Immunity
* Cellular defense mechanism
What type of immunity?
- B cells produce specific antibodies
- Complement binds to immunoglobulin
molecules that have specific complement
receptor sites
Acquired or Adaptive Immunity
* Humoral mechanism
90% of immunohematology testing is done on what type of blood cells?
Red blood cells
-mostly testing for the presence or absence of antigens
Blood group systems are divided based on…
chromosome location or similar location on a gene?
What are the antigen characteristics influencing
immune response?
- Size
- Complexity
- Conformation
- Charge
- Accessibility
- Solubility
- Digestibility
- Chemical composition
What is the second most immunogenetic blood group antigen?
Blood group antigen: D
Blood group system: Rh
Immunogenicity: 50%
You (can or cannot) make an antibody to an antigen you posses?
cannot!
Type of antibodies that are produced without foreign blood exposure. Born with these.
Naturally Occurring Antibodies
type of antibodies:
- Environment stimulated: pollen grains and bacteria membranes
Naturally Occurring Antibodies (check this one)
Which one falls under the naturally occurring antibodies category? and which for the immune antibodies?
IgG and IgM
Naturally Occurring Antibodies- IgM
Immune Antibodies- IgG
type of antibodies that are produced after foreign blood exposure
Immune Antibodies
What are the antibodies that fall under the Immune antibodies category?
Rh, Kell, Duffy, Kidd, and Ss blood group systems
What are the antibodies that fall under the naturally occurring antibodies category?
ABH, Hh, Ii, Lewis, MN, and P blood group systems
React best at 37oC
IgG antibodies
React best at low temp, activate complement, may be hemolytic at 37oC
IgM cold agglutinins
Which is associated with compliment binding? IgG or IgM?
IgG
How many compliment binding sites does IgG have?
2 (small)
What are the heavy chain isotypes?
Gamma- IgG
Alpha- IgA
Mu- IgM
What does the FC region do?
The Fab region?
binds complement
binds antigen
Basic Immunoglobulin Structure
- Papain and pepsin digestion
- Disulfide bonds
- Constant and variable regions
How many antigen binding sited does IgM have?
10 (much bigger than IgG)
What is the light chain type for IgM, IgG, and IgA?
Kappa and Lambda
Fixes complement?
IgM:
IgG:
IgA:
IgM: often
IgG: some
IgA: No
Hemolytic in vitro?
IgM:
IgG:
IgA:
IgM: often
IgG: rare
IgA: No
Placental transfer?
IgG
Placental transfer?
IgM:
IgG:
IgA:
IgM: no
IgG: yes
IgA: no
Direct agglutinin?
IgM:
IgG:
IgA:
IgM: yes
IgG: rare
IgA: rare
Biologic half-life (days)?
IgM:
IgG:
IgA:
IgM: 5
IgG: 21
IgA: 6
majority of IgG is IgG__
1 (65-70)
Which type of IgG has the strongest compliment fixation?
IgG3
Which IgG type is the best at crossing the the placenta?
IgG1
Which IgG type has the shortest biological half life?
IgG3 (7-8days)
-the rest are 21 days
Which IgG type is associated with anti-kidd?
IgG3
Which IgG type is asscociated with anti-Rh, anti-Kell, and anti-Duffy?
IgG1
Which IgG type is associated with anti-platelet?
IgG3
Rh antigens do not bind complement because of…
low levels of antigens present on the RBC surface
Lewis antibodies are generally IgM, but rarely bind complement due to…
low reactivity temperature
Whare are the two most important blood systems to induce complement-mediated intravascular hemolysis?
ABO and Kidds
complement pathways:
Extravascular hemolysis does occur due to…
RBCs coated with antibody
What does the detection of allo- and auto- antibodies depend upon?
binding forces between antigens and antibodies, properties of the
antibody itself, and individual host characteristics.
Antigen-Antibody reactions are influenced by a number of factors, including:
-Distance
* antigen-antibody ratio
* pH
* Temperature
* immunoglobulin type
* Size
* Dosage
* Route
* Degree of foreignness
prozone is __________ excess.
postzone is ________ excess.
what is formed in the zone of equivalence?
antibody, antigen
precipitate (need the right amount of antigen and antibody for the reaction to show)
What are the forces binding antigen to antibody?
-Electrostatic forces (ionic bonding)
-hydrogen bonding
-hydrophobic bonding
-van der Waals forces
The strength of a single antigen-antibody bone produced by the summation
of attractive and repulsive forces
Affinity
The binding strength of a multivalent antigen with antisera produced in an
immunized individual
* Measure of the functional affinity of an antiserum for the whole antigen
* High-titer, low-avidity antibodies = low antigen-binding capacity, but still
reactive at high serum dilutions
Avidity
- Located on RBCs; part of the
cell membrane - Glycoproteins or glycolipids
- Agglutinate with antibodies
- Some antigens can elicit a
stronger immune response
than others (e.g., D antigen)
Antigens
Molecules in plasma or serum
*IgG antibodies are clinically
significant; react at 37° C
*IgM antibodies react at room
temperature or below and are
not usually considered
significant, unless they
activate complement (e.g.,
ABO antibodies)
Antibodies
Antigen-antibody reactions that occur in vitro are detected by visible ____________.
agglutination
What are the 2 steps of Agglutination?
- Sensitization: antibody binds to antigen, but no visible agglutination occurs
- Lattice formation: antibody-coated cells crosslink to form visible agglutination
What are the factors affecting the sensitization of agglutination?
-temperature (IgG-37 degrees, IgM-22 degrees)
-Incubation time
-pH (7 is ideal)
-Ionic strength (adjusted with reagents)
What are the factors affecting the lattice formation in agglutination?
-Zeta potential
-Zone of equivalence
-centrifugation
What are the testing methods used in blood bank?
- Tube testing
- Gel technology
- Solid-phase adherence technology
What are the test systems used in blood blank?
- Immediate Spin (IS) (usually only IgM)
- Antiglobulin Test (AHG phase)
* Direct (DAT)
* Indirect (IAT)
What is always added first when testing in blood bank?
always add “clear” first, non-red cell
What are the steps in the immediate spin test?
-antibody first (clear), then antigen (red cells)
-centrifuge about 10 seconds
-read/interpret
*These are our direct agglutinating antibodies (IgM, room temp)
No red cell button after centrifugation is indicative of….
hemolysis (positive result)
-no red cells means a major reaction
Why is a clump of red cells a negative result on solid phase testing?
When there is nothing to bind to on the well so it collects in the middle instead of being attached to the sides like it would be if it had reacted.
Hemolysis is normally caused by _________ activation.
complement
With Hemolysis reactions, The RBC button is smaller and a pink to red ___________ is observed
after centrifugation
supernatant
- Fresh, nonhemolyzed samples should be used for testing
- Anticoagulants prevent complement activation in vitro by….
chelating calcium
What are the factors that Influence Agglutination Reactions?
-Centrifugation (overcome zeta potential)
-Antigen-Antibody Ratio (prozone, equivalence, postzone)
-pH (6.5-7.5, except anti-M)
-temperature (IS=less than 22, AHG = 37
-Immunoglobulin type (IgM vs. IgG)
What temp is ultra cold?
less than or equal to -65
What temp is frozen?
less than or equal to -18
What temp is refrigerated?
1-6
What temp is room temp?
22-24
What temp is “heat”?
37
Normal saline (0.85-0.9%) can be used with…
IgM antibodies
Examples of enhancement or potentiators that decrease zeta potential (negative charge)?
- AHG – lattice formation
- Protein Media
- 22% Albumin, PEG, Polybrene, PVP, Protamine
- Low Ionic Strength Solution Media: LISS, NHANCE
Examples of proteolytic enzymes used to influence agglutination?
- Ficin (fig) > Papain (papaya) > Bromelin (pinapples) > Trypsin (lining
of hog’s stomach) - Enhances: Rh, Kidd, P1, Lewis, and I
- Destroys: Fya, Fyb, M, N, S, Ch, Rg, Xga
Examples of chemical reduction used to influence agglutination reactions?
- DTT and 2-ME = dissociate IgM but leave IgG intact
- ZZAP = dissociates IgG
What is the affect saline “neat” has on IAT?
60 min incubation
What is the affect 22% Albumin has on IAT
30 min incubation
Allow antibody-coated RBCs
to come into closer contact
with each other “sticky”
What is the advantages and disadvantages of 22% albumin?
adv: none over LISS
dis: cost, long incubation
What is the affect of LISS on IAT?
10-15 min incubation
enhance antibody uptake
What are the advantages of LISS?
adv: shorter incubation
What is the affect of PEG on IAT?
10-15 min incubation
must use anti-IgG
It increases antibody uptake
What are the advantages and disadvantages of PEG?
adv: Increases detection of
clinically significant Abs,
decreases detection of
clinically insignificant Abs
dis: too “sticky”, not as common as LISS
Antihuman globulins obtained from immunized nonhuman species
bind to human globulins such as IgG or Complement
-monospecific
-polyspecific
Antiglobulin Test
* aka Coombs Test
What is the difference between monospecific and polyspecific?
- Monospecific – anti-IgG OR anti-C3b/d (most common)
- Polyspecific – anti-IgG AND C3b/d
this test uses antibody to human antibodies
antiglobulin test aka Coombs test
What is the difference between the way polyclonal and monoclonal anti-human globulins are made?
polyclonal- pooled donor antigen is injected into the rabbit
monoclonal-rabbit is injected with the antigen from one person.
This tests for in vivo sensitization
What is the source for antigen and antibody?
Direct Antiglobulin test (DAT)
red cells source for both
What are the steps in direct antigloubulin test (DAT)?
- Antibody/Antigen (sensitized in vivo)
- Wash (gets rid of free floating antibodies, only way to test attached antibodies)
- Antihuman Globulin (AHG)
- Read/Interpret
When you want to find out if the patient has red cells with antibody already attached, what test is done?
Direct Antiglobulin test (DAT)
What is the control in DAT?
Red cells that are already sensitized (must be positive) check cells???
If negative, wash wasn’t done correctly
What does DAT test for?
-Auto/Alloantibodies
-IgG or Complement
What can cause a DAT test to be positive?
- Neonate due to Hemolytic Disease of the Fetus and Newborn (HDFN)
- Mom’s antibodies attacking baby red cells (father’s antigens)
- Transfused?
- In Vivo hemolysis?
- Serum have unexpected antibodies?
- Receiving drugs?
- Antilymphocyte globulin or antithymocyte globulin
- Intravenous Immune Globulin (IVIG) or Rh Immune Globulin (RhIG)
- Bone marrow or other organ transplant?
What are the steps in the indirect antiglobulin test?
- Antibody + Antigen + Potentiator
- Incubation at 37oC
- Wash (to get rid of unbound IgG)
- Antihuman Globulin (AHG)
- Read/Interpret
What does Indirect Antiglobulin Test (IAT), test for?
- Incomplete Antibodies (in serum, not on RBCs)
* IgG
What is the purpose of adding antibody-coated RBCs to negative reactions?
Checks for neutralization of antisera by free globulin
molecules (Coombs’ control cells are D-positive RBCs coated with anti-D)
DAT vs IAT in detection amounts?
- DAT
- Detects 100-500 IgG
molecules per RBC
* Detects 400-1000 C3d
molecules per RBC
- Detects 100-500 IgG
- IAT
* Detects a minimum of
100-200 IgG or C3
molecules per RBC
What are the sources of error in Antihuman Globulin testing that would give a FALSE POSITIVE result?
- Dirty glassware
- Bacterial contamination of cells or saline
- Saline contaminated by metals or colloidal silica
- Cells with positive DAT will yield a positive IAT
- Polyagglutinable cells
- Overcentrifugation and
overreading - Centrifugation after incubation
phase when PEG is used - Improper specimen (refrigerated, clotted) may cause in vitro
complement attachment - Using serum for DAT
- Using samples collected in gel separator tubes
- Complement attachment when specimens are collected from dextrose infusion lines
- Preservative-dependent antibody directed against reagents
- Presence of fibrin
What are the sources of error in Antihuman Globulin testing that would give a FALSE NEGATIVE result?
- Inadequate or improper washing
- Failure to wash additional times when increase serum is used
- Contamination of AHG
- High concentrations of IgG paraproteins in test serum
- Early dissociation of bound IgG from RBCs
- Interruption in testing
- Improper testing temperature
- Rare antibodies only detectable with polyAHG
- Low saline pH
- AHG reagent nonreactive
- Excessive heat or repeated
freezing/thawing of test serum - Serum nonreactive because of deterioration of complement
- Failure to add AHG, serum, or enhancement medium
***** Under/over-centrifuged - Cell suspension too weak or too heavy
- Serum-to-cell ratios not ideal
- Inadequate incubation conditions in IAT
- Poor reading technique
If positive DAT—> cannot use those cells for IAT, if using same pt. cells?????
?
What immunoglobulin is produced in the primary immune response?
IgM
What immunoglobulin is produced in the secondary immune response?
IgG
Which immunoglobulin is the most efficient at binding complement?
IgM
What portion of the immunoglobulin molecules contains complement binding sites
Heavy chain constant region
What complement pathway is activated by the formation of antigen – antigen complexes?
Classical
Which one of the following properties of antibodies is NOT dependent on the structure of the heavy chain constant region?
a. Ability to cross the placenta
b. Isotype (class)
c. Ability to fix complement
d. Affinity for antigen
Affinity for antigen
Mono clonal, anti-C3d is derived from…
One clone of plasma cells
After the addition of IgG-coated RBCs (check cells) to a negative AHG reaction during an antibody screen, a negative result is observed. Which of the following is a correct interpretation based on these findings?
a. The antibody screen is negative.
b. The antibody screen cannot be interpreted.
c. The saline washings were adequate.
d. AHG reagent was added.
b. The antibody screen cannot be interpreted.
False-positive DAT results are most often associated with:
a. Use of refrigerated, clotted blood samples in which complement components coat RBCs in vitro.
b. A recipient of a recent transfusion manifesting an immune response to recently transfused RBCs.
c. Presence of antispecies antibodies from administration of immune globulin (IVIG).
d. A positive autocontrol caused by polyagglutination.
a. Use of refrigerated, clotted blood samples in which complement
Polyethylene glycol (PEG) enhances antigen-antibody reactions by:
a. Decreasing zeta potential.
b. Concentrating antibody by removing water.
c. Increasing antibody affinity for antigen.
d. Increasing antibody specificity for antigen.
b. Concentrating antibody by removing water.
A positive DAT may be found in which of the following situations?
a. A weak D-positive patient
b. A patient with anti-M
c. HDFN
d. An incompatible crossmatch
C
What do Coombs’ check cells consist of?
a. Type A-positive cells coated with anti-IgG
b. Type A-negative cells coated with anti-IgG
c. Type O-positive cells coated with anti-D
d. Type B-negative cells coated with anti-D
c. Type O-positive cells coated with anti-D
A 27-year-old group O mother has just given birth to a group A baby. Since the mother has IgG anti-A, anti-B and anti-A, B in her plasma, which of the following methods and tests would be most effective at detecting the anti-A on the baby’s RBCs?
a. DAT using common tube technique
b. DAT using gel
c. IAT using common tube technique
d. IAT using gel
A
