Week 10 Part 2 - Cold Agglutinins

Question 1

Benign Cold AIHA

Answer 1

IgM
Usually autoanti-I
Optimally bind to RBCs @ 4°C, may bind up to RT
Relatively low titre (< 64)

Question 2

Pathological Cold AIHA

Answer 2

A.k.a. Cold Agglutinin Disease (CAD)
Acute
- secondary to Mycoplasma pneumoniae infection
Chronic
- elderly, lymphoma, CLL, Waldenstrom’s macroglobulinaemia
IgM antibody
Usually autoanti-I
Wide thermal amplitude
Can react up to 32°C
High titre (>1000)

Question 3

Cold Agglutinin Disease (CAD) Pathogenesis

Answer 3

Antibody binds to RBCs in the peripheral circulation
Complement binds to RBCs
IgM antibody dissociates when RBCs circulate back to warmer areas of the body
Complement remains bound, lyses RBCs

Question 4

CAD Samples in the Lab - Problems and Solutions

Answer 4

Problem - patient RBCs agglutinate in the sample tube (because they react at RT)
Maintain sample @ 37°C until RBCs and plasma are separated
OR
Warm sample for 10-15’ @ 37°C before separating plasma and RBCs
- releases autoadsorbed antibody into plasma
Wash cells with warm saline to remove any residual antibody

Question 5

CAD Samples in the Lab - ABO Discrepancies

Answer 5

May observe ABO discrepancies
- washing cells with warm saline usually resolves discrepant results in the forward reaction
- using prewarmed reagent cells and patient plasma usually resolves discrepant results in the reverse reaction
- use autoadsorbed plasma in individuals with high-titre antibodies

Question 6

CAD Samples in the Lab - DAT Results

Answer 6

DAT +ve with polyspecific (anti-IgG + anti-C3d) reagent, +ve result with only anti-C3d monospecific reagent
IgM antibody therefore no anti-IgG

Question 7

CAD Samples in the Lab - Avoiding Autoantibody

Answer 7

Need to avoid autoantibody masking potential underlying clinically significant alloantibody
Perform IAT @ 37oC using anti-IgG specific reagent (to detect any IgG alloantibodies)

Question 8

CAD Samples in the Lab - Performing an Adsorption

Answer 8

Same rules apply as in WAIHA for performing an auto vs allo absorption in respect to transfusion history
Wash patient cells several times with warm saline to remove bound autoantibody before performing autoadsorption
Perform adsorptions at 4°C

Question 9

How to Differentiate Between Benign and Pathological Cold AIHA - Antibody Titration Studies

Answer 9

Make doubling dilutions of plasma
Add 1 drop of cells to 2 drops of diluted plasma
Incubate at 4oC for 1-2 hrs, centrifuge
Put tubes on ice, read one at a time

Question 10

How to Differentiate Between Benign and Pathological Cold AIHA - Thermal Amplitude Studies

Answer 10

Add 1 drop of cells to 2 drops of patient plasma
Incubate at 25°C, 30°C, 37°C for 1 hr
Check for agglutination

Question 11

Combined/Mixed Type AIHA

Answer 11

Warm IgG autoantibody (IgG) in combination with a cold IgM autoantibody of wide thermal amplitude
IgM antibody is usually lower titre (< 64) compared to CAD
Antibody specificities are as for WAIHA and CAD
DAT +ve with polyspecific and both monospecific (anti-IgG and anti-C3d) reagents
- IgG detected is the warm autoantibody
- C3d remains from cold IgM autoantibody
- eluate is reactive

Question 12

Paroxysmal Cold Haemoglobinuria

Answer 12

Rarest form of AIHA
Acute intravascular HA, secondary to viral infection in children

Question 13

What is Paroxysmal Cold Haemoglobinuria Caused By?

Answer 13

Donath-Landsteiner antibody
- biphasic anti-P IgG antibody that binds complement
- binds to RBCs at lower temperatures (i.e. in peripheral circulation), subsequently binds complement
- aby dissociates when the RBC returns to warmer parts of the body, but complement remains bound, → cell lysis

Question 14

Paroxysmal Cold Haemoglobinuria Other Results

Answer 14

DAT +ve with polyspecific reagent, +ve with only anti-C3d monospecific reagent
Eluate is non-reactive
Antibody is not detected in antibody screen and does not interfere w/ pre-transfusion testing
Detected using Donath-Landsteiner test
Transfuse with cells compatible by IAT