Week 10 - The DAT and Positive Auto-Controls

Question 1

The DAT

Answer 1

Direct Antiglobulin Test, aka Coombs Test
Detects antibody and/or complement that has bound to RBCs in vivo
Performed to differentiate immune from non-immune haemolysis
Performed when the auto-control is positive
Washed patient RBCs are mixed with polyspecific AHG reagent, centrifuged, and checked for agglutination
If performing in tubes, neg reactions should be confirmed w/ sensitised cells (as for IAT)
EDTA plasma is used

Question 2

When is a Positive DAT Seen?

Answer 2

Normal population
- blood donors - 1:1000-1:14 000
- hospital patients - 1:6-1:100
Passively transfused aby from plasma (containing) products
Haemolytic disease of the foetus and newborn
Haemolytic transfusion reaction
Autoimmune haemolytic anaemia

Question 3

Investigation of a Positive DAT

Answer 3

No need to investigate a +ve DAT unless there are signs of haemolysis
Re-perform w/ anti-IgG and anti-C3d monospecific reagents, appropriate diluent control
Perform an elution
Perform antibody identification

Question 4

Elution

Answer 4

Technique used to dissociate antibodies from sensitised RBCs
Use heat, freeze-thaw, acid/EDTA, or organic solvents
Eluate contains concentrated antibody

Question 5

What is an Elution Used For?

Answer 5

Investigation of ABO subgroups and Del
Investigation of HTRs
Investigation of HDNB
Investigation of antibody mixtures (can be used with adsorption to separate antibodies)
Preparation of antibody-free RBCs

Question 6

Auto Control

Answer 6

React patient plasma with patient cells
Not required in antibody screen
Useful in antibody ID

Question 7

When might the Auto Control be Positive?

Answer 7

HTR resulting from a recent transfusion
Autoimmune Haemolytic Anaemia (AIHA)
Antibody against testing medium (i.e. buffer, gel, etc)

Question 8

Positive Auto Control - HTR Resulting from a Recent Transfusion

Answer 8

Recipient produces an alloantibody against an antigen on donor cells
+ve DAT (mixed field)
- antibody binds to donor cells only
- perform an elution, ID antibody in eluate
- if recipient was administered ABO incompatible, plasma- containing component (i.e. platelets), test eluate against A1 and B cells
Might have a negative antibody screen

Question 9

Immune Haemolytic Anaemias

Answer 9

Subject produces antibodies directed against antigens on their
own RBCs
If these antibodies result in shortened RBC survival, → anaemia
Autoimmune haemolytic anaemia (AIHA)
Drug-induced haemolytic anaemias

Question 10

Autoimmune Haemolytic Anaemia Classification

Answer 10

Classified according to the temperatures at which the antibodies are optimally reactive
Warm AIHA
- antibodies are optimally reactive at 37⁰C
Cold AIHA
- antibodies are optimally reactive at <30⁰C
Combined/mixed type AIHA
- mixture of antibodies that react both at 37⁰C and at <30⁰C

Question 11

Warm AIHA

Answer 11

Autoantibody agglutinates all cells tested
- “panagglutinin” and “panagglutination”
- found in plasma and/or eluate
- enhanced by enzymes, PEG, albumin
- antibody may not be detectable in plasma
- no activity in eluate if cells are coated w/ complement only

Question 12

Warm AIHA DAT Results

Answer 12

Rarely DAT negative

Question 13

When Might Warm AIHA Present Problems in Laboratory

Answer 13

Phenotyping
Antibody identification
Selecting blood for transfusion
Cross-matching

Question 14

Warm AIHA Antibody Specificity

Answer 14

Typically, antibody has no apparent specificity (bind to all panel cells)
Occasionally has apparent specificity against Rh antigens (i.e. autoanti-e)

Question 15

When Phenotyping for Warm AIHA, what are the minimum antigens it needs to be tested for?

Answer 15

Rh, K, Duffy (Fy), Kidd (Jk), Ss antigens

Question 16

Warm AIHA Phenotyping

Answer 16

Phenotyping RBCs that are DAT +ve presents problems with conventional reagents
Phenotyping in non-AIHA cases may use IgG antibodies in conjunction with IAT
In AIHA, conventional phenotyping is not possible as the anti-IgG will be bind to the patient autoantibody causeing a false positive

Question 17

Warm AIHA Phenotyping Solution

Answer 17

Elute autoantibody from patient RBCs before phenotyping
Use IgM typing sera
- performed as for an ABO reverse group i.e. 1 drop of cell suspension, 2 drops typing sera, spin, read
- avoids use of anti-IgG used in IAT
RBC genotyping
- used to predict the patient’s phenotype

Question 18

Warm AIHA Alloantibody ID

Answer 18

Autoantibody can mask underlying alloantibodies
- patients can make alloantibodies against antigens that are not expressed on their red cells
Need to avoid interference by autoantibody during antibody ID
- autoantibodies are enhanced by PEG, enzyme-treated RBCs
- low ionic or isotonic saline tube method

Question 19

How to Remove Autoantibody for Warm AIHA

Answer 19

Perform auto adsorption
- incubate patient cells with their own plasma
- autoantibody binds to cells, leaving alloantibody in plasma
- method used depends on transfusion history of patient
If patient has been transfused in last 3 months, can’t perform auto adsorption

Question 20

Why can’t an Auto Adsorption be done if the Patient has been Transfused within the Last 3 Months?

Answer 20

Donor RBCs might adsorb alloantibody
Therefore patient cells cannot be used

Question 21

Test to Perform Instead of Auto Adsorption if Patient has been Transfused within the Last 3 Months?

Answer 21

Allo-adsorption
- use cells other than the patient’s to adsorb the autoantibody from the plasma
- need to ensure that cells which adsorb clinically significant antibodies are NOT used
- cells used depend on whether the phenotype of the patient RBCs is known/can be determined

Question 22

What RBCs should be used for Allo-Adsorption if the Patient’s Phenotype is Known/Not Known

Answer 22

If patient RBC phenotype is known, use cells that best match the patient phenotype for allo-absorption
If patient RBC phenotype is NOT known, use cells negative for those antigens to which clinically significant antibodies could be present
- won’t find a single cell negative for all antigens
- instead, use a panel of antigen negative cells that collectively covers all antigens

Question 23

Warm AIHA - Problems with XM

Answer 23

Focus is to ensure that the presence of underlying clinically significant alloantibodies is excluded
If alloantibodies are present, select antigen negative blood
If patient RBC phenotype is known, select phenotype-matched blood if available
Perform XM using adsorbed plasma