Week 10 - The DAT and Positive Auto-Controls Flashcards
The DAT
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
When is a Positive DAT Seen?
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
Investigation of a Positive DAT
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
Elution
Technique used to dissociate antibodies from sensitised RBCs
Use heat, freeze-thaw, acid/EDTA, or organic solvents
Eluate contains concentrated antibody
What is an Elution Used For?
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
Auto Control
React patient plasma with patient cells
Not required in antibody screen
Useful in antibody ID
When might the Auto Control be Positive?
HTR resulting from a recent transfusion
Autoimmune Haemolytic Anaemia (AIHA)
Antibody against testing medium (i.e. buffer, gel, etc)
Positive Auto Control - HTR Resulting from a Recent Transfusion
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
Immune Haemolytic Anaemias
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
Autoimmune Haemolytic Anaemia Classification
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
Warm AIHA
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
Warm AIHA DAT Results
Rarely DAT negative
When Might Warm AIHA Present Problems in Laboratory
Phenotyping
Antibody identification
Selecting blood for transfusion
Cross-matching
Warm AIHA Antibody Specificity
Typically, antibody has no apparent specificity (bind to all panel cells)
Occasionally has apparent specificity against Rh antigens (i.e. autoanti-e)
When Phenotyping for Warm AIHA, what are the minimum antigens it needs to be tested for?
Rh, K, Duffy (Fy), Kidd (Jk), Ss antigens
Warm AIHA Phenotyping
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
Warm AIHA Phenotyping Solution
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
Warm AIHA Alloantibody ID
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
How to Remove Autoantibody for Warm AIHA
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
Why can’t an Auto Adsorption be done if the Patient has been Transfused within the Last 3 Months?
Donor RBCs might adsorb alloantibody
Therefore patient cells cannot be used
Test to Perform Instead of Auto Adsorption if Patient has been Transfused within the Last 3 Months?
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
What RBCs should be used for Allo-Adsorption if the Patient’s Phenotype is Known/Not Known
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
Warm AIHA - Problems with XM
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
