The direct antiglobulin test (DAT) was first reported in 1908  but found more widespread notoriety after being described in 1945 by Coombs et al. . Fundamentally, the DAT is used to determine whether red blood cells (RBC) have surface bound immunoglobulin G (IgG) and/or complement. The main utility of the DAT is to categorize hemolysis as immune-dependent or immune-independent. This review focuses on clinical utility and the pitfalls of the DAT.
The principle of the DAT is antihuman globulin (AHG) agglutinates, or clumps, antibody-coated cells. Testing typically starts with polyspecific AHG containing both anti-IgG and anti-complement with positive reactions repeated with monospecific AHG to individually detect IgG and complement. Commercial preparations of anti-IgG typically recognize the gamma heavy chain portion of the IgG molecule. Monospecific anti-complement reagents usually contain anti-C3d, although anti-C3b, anti-C4b, and anti-C4d reagents are also available. Binding of anti-C3d is often indicative of, but not limited to, bound IgM . Reagents to identify IgM and IgA heavy chains are not licensed and are not readily available for daily applications.
Several platforms for performing the DAT exist including the conventional test tube (CTT) method and the more sensitive gel microcolumn and solid phase methods (Fig. 1A–C). The DAT is performed using ethylenediaminetetraacetic acid-anticoagulated blood, which inhibits complement binding to RBC in vitro and ensures the detection of only in vivo bound complement . In the CTT method, the patient's RBC are washed with saline to remove unbound immunoglobulin and complement, AHG is added, and the RBC suspension centrifuged. Using gentle tapping, the RBC pellet is dislodged and examined for agglutination, which is graded on a scale from 0 indicating no agglutination to 4+ indicating solid agglutination (Fig. 1A). In the gel microcolumn method, RBC are filtered through a gelatinous matrix mixed with AHG reagents. The gel traps the agglutinated RBC and nonagglutinated RBC pass through the column (Fig. 1B). Solid phase assays use RBC adherence to a well as an endpoint, allowing for results to be interpreted using a spectrophotometer in an automated fashion (Fig. 1C).
Like all tests, the DAT can be falsely negative or positive. Negative reactions are checked for appropriate reagents and reaction conditions by adding sensitized RBC “check cells”; however, this process cannot detect all technical causes of false results. The most common causes for false negative results are improper or under-washing or under-centrifuging the sample that allows residual unbound antibodies to remain in the tube and adsorb the AHG reagent . The failure to add AHG reagents or the addition of inactive AHG reagents due to improper storage and handling may lead to false negative results. A delay in the addition of AHG after washing can also cause false negatives when bound immunoglobulin and complement come off the RBC. In the CTT method, a major cause of false negative results is over agitation at the time of result interpretation . False positive results tend to arise when specimens degrade sufficiently to cause nonspecific binding of the DAT reagents. Causes of false positive results include over-centrifugation which causes the RBC to be packed too tightly, under agitation at the time of result interpretation, a prolonged delay in testing, a clotted specimen, reagent issues, and patient factors such as spontaneous agglutination [4, 5]. It is difficult to say what length of delay in initiating testing or during testing is significant, because the disengagement of antibody from its cognate antigen is dependent upon the unique binding affinity of that complex.
Selected Clinical Applications
The DAT is helpful in the investigation of the cause of suspected hemolysis; however, the results should be interpreted in the context of the clinical situation (Table I).
Table I. Causes of Positive and Negative Direct Antiglobulin Test (DAT) Reactions
2. Autoimmune hemolytic anemia (primary and secondary causes)
a. Warm autoimmune hemolytic anemia
b. Cold autoimmune hemolytic anemia
c. Mixed autoimmune hemolytic anemia
d. Paroxysmal cold hemoglobinuria
3. Transfusion related
a. Acute hemolytic transfusion reaction
b. Delayed hemolytic transfusion reaction
c. Delayed serological reaction
d. Passive transfer of antibody by transfusion
4. Hemolytic disease of the fetus/newborn
5. Passenger lymphocyte syndrome
6. Drug-induced hemolytic anemia
7. Passive transfer of antibody in immunoglobulin preparations
a. Intravenous immune globulin (IVIG)
b. Rh0(D) immune globulin
8. False positive
a. Spontaneous red blood cell agglutination
b. Wharton's jelly in cord blood specimens
i. Poor washing technique
ii. Improper agitation of specimen during reaction strength determination (conventional test tube method)
iv. Clotted specimens
1. Nonimmune causes of hemolysis
2. Drug-induced hemolytic anemia
3. Hemolysis due to an IgA or IgM immunoglobulin
4. Low level of bound antibody and/or complement
5. Low affinity antibody
6. False negative
a. Poor washing technique
b. Improper agitation of specimen during reaction strength determination (conventional test tube method)
c. Failure to add or delayed addition of antihuman globulin (AHG) reagent
d. Inactive antihuman globulin (AHG) reagent
e. Inappropriately concentrated red blood cell suspension
f. Delay in testing
A positive DAT can be found in 1 in 1,000–1:14,000 healthy blood donors without hemolysis [6, 7]. The significance of this finding is unclear, but some individuals may go on to develop autoimmune hemolytic anemia (AIHA) or cancer [8, 9]. The DAT is positive in ∼7–8% of hospitalized patients and up to 15% of hospitalized patient specimens [10 and references therein]. Therefore, the significance of a DAT requires clinical correlation.
Autoimmune hemolytic anemia
AIHA occurs when autoantibodies bind to RBC, causing removal from circulation. AIHA are commonly described as warm (WAIHA) or cold (CAIHA) depending on the optimal reactivity temperature of the autoantibody . WAIHA is the most common form of AIHA. WAIHA is usually caused by IgG autoantibodies that react at 37°C resulting in a DAT positive for IgG. CAIHA is usually caused by IgM autoantibodies that bind to RBC at temperatures between 0°C and 37°C . CAIHA antibodies agglutinate RBC in the peripheral circulation and deposit complement, resulting in a DAT positive for C3d and negative for IgG. Mixed AIHA combines attributes of WAIHA and CAIHA and comprises ∼8% of AIHA . The serum contains IgM autoantibodies with wide temperature reactivity alone or in combination with warm IgG autoantibodies .
Paroxysmal cold hemoglobinuria, the rarest type of AIHA, occurs more often in children [12, 14]. The autoantibody is a biphasic hemolysin that binds RBC at 0°C to 4°C but does not activate complement until warmed to 37°C. The DAT is positive for C3d and negative for IgG. The diagnosis is confirmed by a Donath-Landsteiner test. Testing can be performed on patient blood or patient's serum and reagent RBC with and without the addition of normal serum as a source of complement. The specimens are incubated on melting ice (0°C), at 37°C, and first on ice followed by incubation at 37°C . The specimens are centrifuged and then examined for evidence of hemolysis. A test is positive if hemolysis is detected in the ice followed by incubation at 37°C reaction and not in the melting ice or 37°C reactions.
Transfusion of incompatible RBC results in a hemolytic transfusion reaction (HTR). An acute HTR (AHTR) occurs within 24 hr of a transfusion and is due to pre-existing antibodies binding to RBC resulting in hemolysis. ABO incompatibility is the most common cause of fatal AHTR; however, antibodies to Kell, Kidd, Duffy, and other RBC antigens have been reported . A delayed HTR (DHTR) occurs greater than 24 hr post-transfusion and may present with unexplained anemia. De novo production of a RBC-specific antibody or an anamnestic response of a previously undetectable antibody may both cause DHTR. The DAT may be positive for immunoglobulin and/or complement; however, a negative DAT does not exclude an HTR as antibody-bound RBC may have been rapidly cleared from circulation.
HTRs also occur with passive transfer of antibody. For example, blood type B platelets containing anti-A in the plasma may react with recipient RBC when transfused to a blood type A patient. Using components with low titers of antibodies or removing the antibodies by washing may lower this risk.
Hemolytic disease of the fetus/newborn
Hemolytic disease of the fetus/newborn (HDFN) causes a positive DAT in neonates because maternal IgG antibodies with specificity for fetal RBC antigens cross the placenta, bind fetal RBC, and cause hemolysis. Antibodies to ABO antigens are the most common cause of mild HDFN . Rh and Kell antibodies are the next most common cause of HDFN and tend to cause more severe disease, but antibodies to many other RBC antigens including anti- Kidd, Duffy, MNS, and Diego have also been implicated . A positive DAT in a neonate needs correlation with ABO type and antibody screen results of the patient and mother and Rh0(D) immune globulin administration to the mother. Wharton's jelly contamination of a cord blood specimen can result in a false positive DAT .
Drug induced hemolytic anemia
Drug-induced hemolytic anemia (DIHA) is rare, occurring in an estimated 1 in 1 million individuals . The true incidence is likely higher as mild cases go unrecognized . Beta-lactam antimicrobials such as cephalosporins or penicillin and its derivatives, nonsteroidal anti-inflammatory drugs, and quinine/quinidine are common offenders [21, 22]. DIHA may have a positive DAT for IgG and/or C3 with variable reactivity or a negative DAT depending on the mechanism leading to hemolysis [22, 23]. Reference laboratory testing in the presence and absence of the suspected drug may confirm the diagnosis. DIHA is difficult to distinguish from AIHA and may not be clarified until the drug is discontinued.
Drugs containing monoclonal or polyclonal antibodies may directly bind to RBC causing potential hemolysis and a positive DAT. Two examples include intravenous immunoglobulin, a polyclonal antibody mixture generated from large donor pools, and Rh0(D) immune globulin, which is used in idiopathic thrombocytopenic purpura and prevention of HDFN.
Passenger lymphocyte syndrome
Passenger lymphocyte syndrome occurs when B-lymphocytes transplanted along with an allograft generate antibodies toward host RBC antigens. Most cases are due to ABO incompatibility; however, rare cases are attributed to other blood groups such as Rh, Kidd, Kell, and Duffy [24–26]. Hemolysis typically begins 7–14 days post transplant and lasts 5–10 days with rare cases having persistent evidence of antibody production and hemolysis .
DAT-negative immune mediated hemolysis
Despite an immune mechanism for hemolysis, the DAT may be negative for several reasons in addition to those described above. Autoantibodies of the IgA or IgM subtypes may cause hemolysis with a negative DAT because the test is designed to only detect RBC coated with IgG or C3d [11, 27]. The DAT may not have adequate sensitivity to detect low levels of potent antibodies . Clinically relevant low affinity antibodies may be removed by washing during the DAT testing . Other technical issues may lead to false negative results. In the setting of a negative DAT, nonimmune causes for hemolysis should also be considered.
There are multiple etiologies of hemolysis. The DAT is invaluable for classifying the cause of RBC destruction as immune or nonimmune, although some disorders may not be correctly classified. Therefore, the DAT results must be interpreted in the context of the clinical situation. Several methods for performing the DAT are available, and clinicians using the DAT need to be familiar with each testing methodology's sensitivity, specificity, pitfalls, and clinically relevant correlates of testing results.