Introduction

Antiglobulin testing, also known as the Coombs test, is an immunology laboratory procedure used to detect the presence of antibodies against circulating red blood cells (RBCs) in the body, which induce hemolysis. The destruction of these red blood cells (RBCs) by antibodies directed against them is described diagnostically as autoimmune hemolytic anemia (AIHA). Many etiologies fall under this classification.

Antiglobulin testing can be either direct antiglobulin testing (DAT) or indirect antiglobulin testing (IAT). The principle of DAT is to detect the presence of antibodies attached directly to the RBCs, which takes place by washing a collected blood sample in saline to isolate the patient’s RBCs; this procedure removes unbound antibodies that may otherwise confound the result. IAT, by contrast, is used to detect unbound antibodies to RBCs, which may be present in the patient’s serum. Direct antiglobulin testing adds a monospecific or polyspecific reagent to the washed RBCs to detect bound IgG and/or complement C3. In practice, many laboratories first use the polyspecific reagent that can detect both IgG and C3; a positive result is followed by monospecific testing to characterize the antibody further.[1] For indirect antiglobulin testing, serum from a blood sample gets isolated, and native RBCs are removed. The isolated serum sample then gets incubated with foreign RBCs of known antigenicity. Antiglobulin reagent is then added, and the presence of agglutination indicates a positive result.

Specimen Collection

Collecting a blood sample for antiglobulin testing requires an anticoagulated tube with ethylenediaminetetraacetic acid (EDTA); in standard practice, this collection tube traditionally has a lavender, red, or pink top. EDTA is used to chelate serum calcium to prevent in vitro fixation of complement factor C3, which would otherwise lead to a false negative result.[1]

Procedures

Various modifications have been reported to improve the Coombs test, including polyethylene glycol (PEG) and the antiglobulin gel test (AGT).[2][3] Some advantages of the AGT compared to the standard Coombs test include better reproducibility and easy testing. The AGT is the most sensitive test for detecting anti-RBC antibodies in the serum. It is essential in pre-transfusion testing and the diagnosis of hemolytic disease in the newborn.[4] The AGT was released in Europe in 1988. It became available in the USA in 1995. Lapierre and collaborators developed this technology.

The AGT is routinely used to screen human sera for anti-human RBC antibodies. The procedure is as follows:

• Use a gel microtube (it contains anti-IgG)
• Add 25 microliters of serum to the microtube
• Add 50 microliters of low ionic strength solution (LISS) - suspended red blood cells at a 0.8% concentration to the reaction chamber of the microtube
• Incubate at 37 degrees Celcius for 15 minutes
• Spin for 10 minutes in a centrifuge at approximately 70 x g
• After centrifugation, the positive reaction gets graded from 0 to 4+
• Negative reactions have RBC pellets on the bottom of the microtube with no agglutination
• One + reaction is indicated by erythrocyte agglutination at the lower half of the gel column
• Two + reaction has erythrocytes dispersed throughout the microtube
• Three + reaction contains erythrocytes displayed in the upper half of the gel column
• Four + reaction is indicated by a solid band of erythrocyte on the top of the microtube chamber [5] 

Indications

Coombs test is necessary when autoimmunity to red blood cells is a consideration in the differential diagnosis, including warm and cold hemolytic anemia. Following are some indications where antiglobulin testing becomes useful:

• Autoimmune hemolytic anemia

• Drug-induced immune hemolytic anemia

• Alloantibodies-mediated hemolytic transfusion reactions

• Hemolytic disease of the newborn

• Systemic lupus erythematosus (without hemolytic anemia)

Potential Diagnosis

The potential diagnosis of the Coombs test includes pre-transfusion testing, hemolytic transfusion reaction, and autoimmune or drug-induced hemolytic anemias.[6][7] There are several causes of a positive Coombs test, such as:

• Hemolytic transfusion reactions

• Autoantibodies to intrinsic RBC antigens

• Hemolytic disease of the newborn

• Drug-induced antibodies

• Passively acquired alloantibodies, such as from donor plasma or immunoglobulin

• Nonspecifically adsorbed proteins

• Complement activation because of bacterial infection, alloantibodies, or autoantibodies

• Antibodies produced by passenger lymphocytes

Normal and Critical Findings

Reporting of antiglobulin agglutination test results can be on a qualitative or quantitative basis. For qualitative methods, the interpreter examines the test tube and assigns a score based on a graded scale:

• M: Mixed field – any degree of agglutination in a sea of non-agglutinated cells
• W: Siny aggregates, turbid reddish background
• 1: Small aggregates, turbid reddish background
• 2: Small to medium-sized aggregates, clear background
• 3: Several large aggregates, clear background
• 4: Aggregate or clump of cells

In patients with autoimmune hemolytic anemia, the degree of agglutination typically correlates with the severity of hemolysis. If no macroscopic agglutination appears, the sample is examined microscopically to ensure no aggregates. A sample exposed to a reagent demonstrating aggregates of at least 3 to 5 cells under microscopic examination is considered a positive result. Agglutination typically takes 5 to 10 minutes after adding the reagent. Direct antiglobulin testing may also be measured quantitatively using enzyme-linked immunosorbent assay (ELISA), flow cytometry, or other immunoassay techniques.[8][9][10][11] Quantitative sample measurement may be necessary when isolation of a specific antibody is desired, such as in cases of autoimmune hemolysis due to antibodies other than IgG or C3.

Interfering Factors

When performed correctly and utilized in the appropriate clinical context, direct antiglobulin testing has been shown to demonstrate a positive predictive value of 97% to 99%, although a more recent study involving hospitalized patients reported a false positive rate of up to 7% to 8% in patients without any evidence of hemolysis clinically or histologically. The majority of these false positives show a low grade of agglutination, though up to 1% of these results may demonstrate higher grades of agglutination.[12] In another study consisting of a cohort of healthy, non-hospitalized individuals, the incidence of a positive DAT result without evidence of hemolysis was found to be 0.1%; approximately two-thirds of this cohort expressed IgG positivity.[13]

Several other confounding variables can affect the accuracy of DAT and IAT test results:

• Type of antibody - most commercial antiglobulin testing screens for antibodies to IgG, complement C3, or both. As such, false-negative results may occur in cases of AIHA caused by autoantibodies other than IgG or C3, such as IgM or IgA.[14] In these uncommon cases, quantitative DAT may aid in detection.
• Amount of antibody present - in some rare instances, autoimmune anemia may be induced by antibody levels below the detection limit of DAT, which is approximately 150 to 500 molecules of IgG per red cell.[15][16] For example, the study conducted by Zupanska et al demonstrated in vitro phagocytosis of RBCs by monocytes at levels of 150 to 640 molecules of IgG3 and 1230 to 4240 molecules of IgG1 per red cell.[17][18] Thus, RBCs may undergo phagocytosis when opsonized by an antibody level below the detection threshold, resulting in a falsely negative result.[12]
• High serum protein - certain diseases, such as myeloproliferative diseases, may cause a falsely positive agglutination study due to abnormally high protein levels unrelated to antibody-RBC agglutination. Exogenous sources of excess protein or immunoglobulin, such as cases in which a patient receives intravenous immune globulin (IVIG), may also result in a false positive study.
• Infection - the serum of individuals infected with certain microorganisms may create a false positive agglutination result. Examples include human immunodeficiency virus (HIV), malaria, hepatitis C virus (HCV), and in rare cases, the hepatitis E virus (HEV).[19]
• Antiphospholipid syndrome - cross-reactivity between antiphospholipid antibodies and RBC membranes can result in falsely positive DAT testing, as reported by Win et al [20]
• Wharton jelly - in neonatal umbilical cord blood samples, mucopolysaccharide-rich Wharton jelly has been shown to produce false-positive antiglobulin results.

Patient Safety and Education

The patient should be aware that Coombs testing is a relatively safe procedure. The risks associated with testing are the same as that of standard blood sample collection. Patients do not need to fast before testing.

Clinical Significance

Antiglobulin testing, particularly qualitative direct antiglobulin testing, is clinically useful in cases where there is clinical suspicion of autoantibody-induced RBC hemolysis. DAT testing typically involves using a polyspecific reagent consisting of IgG and complement C3. Indirect antiglobulin testing is clinically useful for the detection of circulating antibodies that have the potential to induce RBC hemolysis; this test is most commonly utilized for RBC phenotyping and in crossmatch screening for blood transfusion. A positive antiglobulin result requires analysis in the clinical context to make an accurate diagnosis. Healthcare costs and the burden of laboratory time can be minimized by screening with a polyspecific reagent before confirming the antibody with monospecific or quantitative analysis. Rarely, autoimmune hemolysis may be suspected even without positive DAT testing; in this instance, quantitative DAT testing may help identify less common antibody subtypes other than IgG or C3. In the absence of other confounding variables (see “Interfering Factors” above), positive antiglobulin testing indicates the presence of hemolysis by antibodies directed against native RBCs. There are several major areas of clinical significance:

• Autoimmune hemolytic anemia (AIHA): AIHA is traditionally the most recognized cause of positive antiglobulin testing, and has been the topic of extensive study. The classification “AIHA” serves as an overarching descriptor that unifies a large group of diagnoses with differing etiologies that cause hemolysis using antibodies against RBCs.[21] The classification can be dichotomized further by considering factors such as warm versus cold agglutination and primary versus secondary cause. AIHA may also be drug-induced or syndromic (see “Evans syndrome”). Further characterization of diseases that fall under this classification is beyond the scope of this entry.

• Alloimmune-mediated hemolytic transfusion reaction (AHTR): AHTR occurs when a post-transfusion specimen develops a newly found alloantibody. The formation of an alloantibody can occur as quickly as within 2 to 3 days.[22] The development of alloantibodies results in a positive DAT test but may or may not be associated with hemolysis.[1]

• ABO blood group typing: In blood transfusions and hematopoietic stem cell transplants, indirect antiglobulin testing can be used to identify the RBC phenotype to minimize the chances of donor incompatibility.

• Hemolytic disease of the fetus and the newborn (HDFN): HDFN occurs when maternal IgG forms against fetal antigens, notably the Rh or Kell antigen. The most common type of HDFN is due to ABO incompatibility, which occurs in approximately 15% to 25% of pregnancies and tends to be less severe.[23] The incidence of positive DAT testing in ABO HDFN is very low at around 1%, and of that group, only approximately 23% of newborns develop clinically significant jaundice; hence, DAT is a poor positive predictor of newborns that require treatment.[1][23][24]