Tests of haemolysis - direct antiglobulin test

What it detects

*Detects IgG and/or complement (C3d) already bound in vivo to the patient's red cells*
The test that separates immune from non-immune haemolysis - nothing else does

	Direct (DAT / direct Coombs)	Indirect (IAT)
Sample	Patient's red CELLS	Patient's PLASMA
Detects	Antibody/complement already on the cells	Free antibody in the plasma
Uses	AIHA, HDFN, haemolytic transfusion reaction, drug-induced immune haemolysis	Antibody screen, crossmatch, antenatal screening

Method: wash the patient's red cells, add anti-human globulin (Coombs reagent) -> agglutination if antibody or complement is bound. Monospecific reagents (anti-IgG, anti-C3d) identify which

Positive in ~1 in 1,000-10,000 healthy blood donors - a positive DAT is far more common than autoimmune haemolysis
Positive in ~5-15% of hospitalised patients, most without haemolysis
~5-10% of genuine warm AIHA is DAT-negative

Pattern	Diagnosis
IgG only	Warm AIHA, drug-induced (hapten/autoantibody type), HDFN
IgG + C3d	Warm AIHA - more complement fixation, often more severe
*C3d only*	*Cold agglutinin disease, paroxysmal cold haemoglobinuria, some drug-induced immune complex haemolysis. ~5% of warm AIHA*
Negative	DAT-negative AIHA, hereditary/enzymatic/mechanical haemolysis, PNH

Destruction requires the antibody to be recognised - splenic macrophage Fc-gamma receptors bind IgG1 and IgG3 far more efficiently than IgG2 or IgG4, and IgG1/IgG3 are the subclasses that fix complement effectively
- -> a DAT positive for IgG2 or IgG4 alone may cause no haemolysis at all
Antibody density matters - below a threshold of molecules per cell, clearance does not occur
Thermal amplitude matters - a cold antibody that only binds at 4 C is clinically irrelevant
*Therefore: a positive DAT is a laboratory finding. Haemolysis is a clinical and biochemical diagnosis.*

Antibody density below the assay threshold (<~200-500 IgG molecules per cell)
Low-affinity IgG washed off during the procedure
IgA- or IgM-only autoantibodies - not detected by standard anti-IgG/anti-C3d reagents
Specialised techniques - flow cytometry, column agglutination, microcolumn or enzyme-linked DAT - detect these

Any new haemolytic anaemia: high LDH, high unconjugated bilirubin, low haptoglobin, high reticulocytes
Suspected haemolytic transfusion reaction
Neonatal jaundice or anaemia (HDFN)
Unexplained anaemia in CLL, lymphoma, SLE, post-transplant
Before attributing haemolysis to a drug

Blood film (spherocytes -> warm AIHA; agglutination -> cold; schistocytes -> TMA, and the DAT will be negative)
Reticulocytes, LDH, bilirubin, haptoglobin
Recent transfusion history (mixed-field DAT positivity = delayed haemolytic transfusion reaction)
Drug history - methyldopa, penicillins, cefotetan, ceftriaxone, quinine, fludarabine, checkpoint inhibitors
Recent IVIG, anti-D, ATG or daratumumab - all can cause a positive DAT without haemolysis

Hypergammaglobulinaemia - myeloma, HIV, chronic infection, autoimmune disease
IVIG and intravenous anti-D
Daratumumab - binds CD38 on red cells; causes pan-reactivity that interferes with crossmatching for months. Baseline extended phenotype before starting anti-CD38 therapy**
Recent transfusion, ABO-mismatched transplant, solid organ transplant (passenger lymphocytes)
Sepsis, renal failure, sickle cell disease, elderly patients

Acid or heat elution removes bound antibody from the cells so it can be identified
Distinguishes alloantibody (delayed transfusion reaction) from autoantibody (AIHA)

Confirms immune-mediated haemolysis when haemolysis is biochemically present -> treat as AIHA and search for a secondary cause (CLL/lymphoma, SLE, HIV, drug)
In a transfusion reaction -> investigate for haemolytic reaction, return the unit, repeat group and screen
In a neonate -> HDFN; monitor bilirubin, consider phototherapy/exchange

*A positive DAT in the absence of haemolysis needs no treatment* - no steroid, no further investigation beyond an explanation
*A negative DAT does not exclude AIHA* - if the clinical picture fits (spherocytes, response to steroid, no other cause), refer for specialist DAT techniques

A warm autoantibody makes every unit "incompatible" on crossmatch
- Do not withhold necessary transfusion
- Transfusion medicine performs adsorption studies to exclude an underlying alloantibody, then issues phenotype-matched "least incompatible" units
*Take a baseline extended red cell phenotype or genotype before starting daratumumab, rituximab-containing regimens or chronic transfusion*

Warm and cold autoimmune haemolytic anaemia; Evans syndrome
CLL, non-Hodgkin lymphoma, Hodgkin lymphoma, Waldenstrom macroglobulinaemia
SLE, RA, autoimmune hepatitis, common variable immunodeficiency, ALPS
Haemolytic disease of the fetus and newborn
Acute and delayed haemolytic transfusion reactions; hyperhaemolysis in sickle cell disease
Drugs - methyldopa, penicillins, cefotetan, ceftriaxone, quinine, fludarabine, immune checkpoint inhibitors
Daratumumab and other anti-CD38 antibodies (interference, not haemolysis)
Pneumococcal HUS (T-antigen exposure - the one HUS with a positive DAT)
Post-allogeneic transplant, solid organ transplant (passenger lymphocyte syndrome)

The DAT usually remains positive for weeks to months after successful treatment of AIHA - it is not a response marker; use haemoglobin, reticulocytes and LDH instead
A drug-induced positive DAT can persist for months after the drug is stopped (classically methyldopa)
Daratumumab interference persists for up to ~6 months after the last dose

*Positive DAT without haemolysis is common and needs no action*
*Negative DAT does not exclude AIHA*
*A positive DAT in a TMA points away from HUS/TTP and towards an alternative diagnosis* (except pneumococcal HUS)
Repeat testing adds nothing once the diagnosis is established

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