W9TL1 - RBC Immunology and Transfusion Techniques

Question 1

RBC Antigen Immunogenicity

Answer 1

D - 50%
K - 5%
c - ~2%
E - ~1.7%
k - 1.5%
e, Fya, C, JKa, S, JKb, s - each is < 1%

Question 2

Consequences of Antigen/Antibody Binding

Answer 2

In vivo, an antibody can bind to a RBC antigen
RBC is damaged via
- complement activation
- antibody-dependent cell-mediated cytotoxicity (ADCC)
Results in intravascular or extravascular haemolysis, which = BAD!!
- haemolytic transfusion reaction (HTR)
- haemolytic disease of the newborn (HDN)

Question 3

Clinically Significant Antibodies

Answer 3

Cause an adverse transfusion reaction when RBCs expressing the corresponding antigen are transfused
IgM
- ABO, Lewis, MN from the MNS group, P, I blood group antibodies
IgG
- Rhesus, Kell, Kidd, Duffy, SsU from the MNS group blood group antibodies

Question 4

Antibody Detection Methods - Immediate Spin

Answer 4

Add patient plasma to donor/known cells, spin, read
Performed at RT
Detects IgM antibodies

Question 5

Forward and Reverse Grouping

Answer 5

Forward - react patient cells with antisera

Reverse - react known cells with plasma

Question 6

Antiglobulin Tests - IAT

Answer 6

Used in antibody screening/identification and XM’s
Add patient plasma to donor/panel cells, incubate, WASH, add AHG
reagent, spin, read
Detects IgM at IS, strong IgG at 37°C and IgG at AHG

Question 7

Antiglobulin Tests - DAT

Answer 7

Detects aby (+/- complement) bound to RBCs in vivo
- HTR, HDNB, autoimmune haemolytic anaemias (AIHA)
WASH patient cells, add AHG reagent, incubate, spin, read
Detects IgG antibodies or complement proteins bound to RBCs

Question 8

What is the major difference between the IAT and DAT

Answer 8

DAT detects IgG complement that bind to RBCs IN VIVO
Whereas the IAT detects these components that bind in VITRO
Therefore, when performing the DAT, we test only the patient cells, because the source of antibody and/or complement is that already bound to the patient cells

Question 9

IAT Controls

Answer 9

Add AHG control (sensitised cells) to negative reactions
RBCs with antibody bound to surface
Binds free AHG in solution
If still no agglutination after addition of controls, the results are considered invalid

Question 10

Column Agglutination Technology

Answer 10

Plastic column contains glass or Sephadex beads/matrix
Reagent antibody can be incorporated into the bead
High density solution is laid over top of bead
- traps plasma proteins
- AHG tests don’t require washing!!!!
Use 0.8-1% cell suspension
50 μL cells (+/- 50 μL plasma) are added into the top of the column
Card is centrifuged
Agglutinated cells are trapped at the top of the gel matrix
Cells not agglutinated can move through the gel and form a button at the bottom of the column

Question 11

Column Agglutination Technology - The Control Tube

Answer 11

The control tube on cards contains the antibody diluent
It should be negative
If positive, agglutination in other tubes must be disregarded
Causes of false positives:
- RBC concentration is too high
- expired card
- improper centrifugation
- fibrin in sample
- lipaemic samples
- rouleaux
- Wharton’s Jelly

Question 12

CAT vs Tubes

Answer 12

Advantages of CAT:
- standardisation in reading reactions
- increased sensitivity
- results are stable for ~24 hrs
- washing is not required
- AHG control cells are not required
- reaction setup and card reading can be automated
Disadvantages of CAT
- ABO/D grouping card cost ~$5 each
- IAT card cost ~$10 each
- equipment – centrifuges, incubators, pipettes, etc.

Question 13

Solid Phase Red Cell Adherance

Answer 13

Start with U-bottomed microwell
RBCs are added to the microwell then lysed
LISS and plasma are added to the well
Antibodies against RBC antigens bind to the lysed RBCs
Well is washed
Add indicator cells (RBCs coated with anti-IgG antibody)
Centrifuge
Positive reaction - indicator RBCs cover the microwell surface
Negative reaction - Indicator RBCs are found as a button at the bottom of the well

Question 14

SPRCA vs tube/CAT

Answer 14

Advantages:
- detects only IgG antibodies
- increased sensitivity
Disadvantages:
- detects non-specific RBC antibodies

Question 15

What antibodies are primarily produced in the primary and secondary reactions and what is the secondary reaction called in transfusion?

Answer 15

Primary response - IgM
Secondary response - IgG
The secondary response is known as the Anamnestic response