Practical 7: Crossmatch

Question 1

What is the principle behind the antiglobulin test?

Answer 1

Commercial antibody with a specificity toward human
globulins is used to agglutinate antibody-coated (or complement) RBCs

Reagent contains antibodies toward:
* IgG (anti-IgG) and/or
* Complement (anti-C3d, anti-C3b)

Question 2

What’s the principle and procedure behind the indirect AHG
(3)

Answer 2

Detects IgG or complement bound to RBCs

Two-step procedure:
* Antibodies (in serum) are incubated at 37° C with RBC
antigens in vitro

• RBC suspension is washed and then combined with AHG reagent to detect agglutination

Question 3

What are the uses for IAT
(4)

Answer 3

Antibody screening
Antibody identification
Crossmatching
Antigen typing

Question 4

What is the purpose for crossmatching?

Answer 4

Determines serologic compatibility between donor and patient before transfusion

Question 5

What happens if washing goes wrong in IAT?

Answer 5

Free IgG remains and interferes with AHG binding to Red Cell bound IgG causing a
False Negative as all AHG bound to free IgG and no excess to crosslink coated cells

Question 6

What should you do if you have negative crossmatches with the indirect IAT?

Answer 6

Add 2 drops IgG Sensitised (a.k.a. Coombs Control Cells) and Centrifuge P5 Read Macro

Should be positive

Question 7

What is your positive control for IAT in gels

Answer 7

Weak Anti-D (25ul) + an (50ul) ORhD Pos Cel

Question 8

What is your negative control for IAT in gels

Answer 8

AB Serum (25ul) + an (50ul) ORhD Pos Cell

Question 9

What does the DAT do?

Answer 9

Detects IgG or complement bound to RBCs
● In the procedure, AHG reagent is added after the RBCs
have been washed
●Agglutination demonstrates whether IgG or
complement was attached to the RBCs

Question 10

What might cause positive DAT

Answer 10

Transfusion reaction
Haemolytic disease of the newborn
Autoimmune haemolytic anaemia
Drug-related mechanism

Question 11

What factors affect the IAT

Answer 11

Ratio of serum to cells
Temperature
Reaction medium: LISS
Incubation time

Question 12

What are the most common sources of error for the IAT

Answer 12

Inadequate washing

Nonreactive AHG reagent

Failure to add AHG reagent
(Green Colour helps)

All negative antiglobulin test
reactions must be checked by
the addition of IgG-sensitized
cells