Principle of Hemagglutination and Testing Platforms in the Blood Bank

Of the tests to be discussed later, a basic tenet inherent to all is mixing patient specimen (plasma or RBCs) with a reagent to examine for a reaction. Positive testing in the blood bank setting typically is reflected as hemagglutination. The strength of the agglutination reaction is graded on a 0 to 4+ scale, with 0 indicating a negative test (ie, no agglutination) and 4+ indicating an overwhelmingly positive reaction.

Hemagglutination can take place via several laboratory platforms. Historically, compatibility testing was performed in glass or plastic tubes, with patient specimen and reagent added, and, after several incubation and wash steps, observation for agglutination. Given the moderate sensitivity and laborious nature of the tube method, several technological advancements were subsequently introduced. Although still based on the fundamental principle of agglutination, platforms such as automated gel columns have been developed to further augment a blood bank’s ability to detect RBC antibodies. More recently, automated platforms involving solid phase RBC adherence (wherein a patient’s plasma is mixed with immobilized RBC antigens on a plate or well) have been accepted into clinical practice. Although gel and solid phase platforms have increased sensitivity compared with tube methods, this comes with a concomitant increase in false-positive reactions.

ABO and Rh(D) Blood Group Phenotyping

The ABO system, representing a series of carbohydrate antigens on the RBC surface, is the single most important blood group family for transfusion compatibility. Infusion of ABO incompatible blood components can result in severe hemolytic reactions, morbidity, and death. ABO antigens are particularly problematic because individuals lacking a given antigen within this system make a corresponding antibody from early in life without the need for prior RBC exposure via transfusion or pregnancy. As such, thorough and reliable mechanisms for determining an individual’s ABO status are critical for compatibility.

Blood banks typically perform 2 steps in determination of an individual’s ABO status: forward grouping for the presence or absence of A or B antigens (informally referred to as the front type) and reverse grouping for the presence of anti-A or anti-B antibodies in plasma (informally referred to as the back type). The front type involves detection of A or B antigen using reagent antibodies directed against these targets. The back type is meant to serve as a confirmation of the results of the front type and is based on the previously described principle that an individual lacking a given ABO antigen should have a detectable antibody against that antigen.

In terms of clinical significance, the Rh blood group antigen system is the second most important after ABO. Unlike ABO antigens, which are carbohydrate in nature, Rh antigens are polypeptide and typically require a previous exposure before induction of an alloantibody. Also unlike the ABO system, the Rh system is highly complex with more than 30 unique antigens. However, during routine practice, blood banks only test for the presence or absence of a single antigen in the Rh system: Rh(D).

Phenotyping for the other, non-Rh(D), minor blood group antigens may also be completed for select patient populations. Such phenotyping for antigens including C/c, E/e, and K is also typically done by mixing patient RBCs with antisera against these antigens and can be completed with tube or automated methods. As described in more detail later, determining an extended antigen phenotype is typically reserved for patients at high risk for RBC alloimmunization.

Indirect Antiglobulin Test

The type and screen is the most frequently performed test in the blood bank. When providers order a screen, blood banks are performing an indirect antiglobulin test (IAT) to identify possible autoantibodies or alloantibodies in patients’ plasma. Fig. 1 provides an overview of the IAT, which involves mixture of patient plasma with screening cells expressing various combinations of clinically relevant, non-ABO antigens. IATs are designed primarily to detect immunoglobulin (Ig) G class antibodies. If an IAT is negative, then there is no evidence for an existing autoantibody or alloantibody in the patient’s plasma and blood banks will provide RBCs for transfusion. However, if an IAT is positive, blood banks must identify the cause of that positive reaction such that compatible units for RBC transfusion can be provided. As such, blood banks reflex positive IATs to RBC antibody panel testing.

The IAT. This assay is used primarily for the detection of IgG class autoantibodies and/or alloantibodies in patient plasma. It involves a mixture of a patient’s plasma with reagent screening cells (SCs) expressing a variety of blood group antigens, followed by the addition of anti–human globulin (AHG). A positive IAT manifests as an agglutination reaction.

Identification of Red Blood Cell Antibodies by Panel Testing

The panel assay is performed using a mixture of patient plasma and numerous reagent cell lines. In many ways, panel testing is analogous to the simple IAT screen, with the most significant difference being the number of reagent cells used (10–12 in the panel vs 2–4 in the screen). Selection of a larger number of reagent cells in the panel helps blood banks more clearly identify the antibodies driving the positive reaction.

Note that not every positive IAT reaction is attributable to an alloantibody. Warm-reactive autoantibodies frequently give rise to positive screening tests. However, compared with the results seen with an alloantibody, panels for patients with autoantibodies generally show panagglutination; that is, reactivity with every reagent cell tested. Such a finding typically warrants the performance of a direct antiglobulin test (DAT).

Direct Antiglobulin Test

The DAT is a highly sensitive assay used to detect antibody or complement bound to the RBC surface. As shown in Fig. 2 , DATs are typically 2-step tests, with the first step involving a polyspecific reagent detecting either IgG or complement component C3 on the RBC surface. Agglutination with polyspecific reagents requires additional testing to determine whether IgG, C3, or both are on the RBC surface.

The DAT. This assay is used primarily for the determination of whether IgG or complement component C3 is bound to a patient’s RBCs; the primary end point is agglutination. The DAT is a 2-part test with the first step involving a mixture of patient’s specimen with a polyspecific reagent capable of agglutinating either IgG and/or C3-coated RBCs. If the polyspecific step shows RBC agglutination, then specific testing using AHG or anti-C3 must be used to determine whether IgG, C3, or both are coating the patient’s RBCs.

A positive DAT with IgG or C3 may be suggestive of a hemolytic picture, with IgG reactivity often correlating with a warm-reactive antibody/extravascular hemolysis and C3 reactivity typically correlating with a cold-reactive antibody/intravascular hemolysis. However, as a highly sensitive test, the DAT can also be prone to false-positive reactions. Causes of false-positive DATs (ie, agglutination in the absence of immune-mediated hemolysis) include the following:

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  Administration of drugs that modify the RBC surface (eg, antibiotics)

  
  
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  Polyclonal hypergammaglobulinemia

  
  
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  Previous transfusions

  
  
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  Severe RBC rouleaux

Red Blood Cell Elution

RBC elution is used to evaluate the specificity of an antibody bound to the RBC surface. Although several laboratory techniques are available to help liberate antibodies from RBCs, many blood banks use an acid elution approach for optimal removal. As outlined in Fig. 3 , the interaction between RBC and antibody is weakened in an acidic environment, allowing formerly bound RBC antibodies to accumulate in the plasma. Specimens are then centrifuged and the supernatant plasma removed. The antibody-concentrated supernatant is then reacted with an RBC panel to identify the antibody specificity.

RBC elution studies are used to evaluate a positive DAT showing IgG reactivity. In this assay, a patient’s antibody-coated RBCs are treated with a buffer that causes dissociation of the antibodies from the RBC surface. This concentrated RBC eluate is then reacted against reagent panel cells to determine the specificity of the autoantibodies or alloantibodies.

In the setting of AIHA, in which autoantibodies are targeting highly conserved portions of the RBC membrane, RBC elution studies show panagglutination. In contrast, in the setting of alloimmune hemolysis (eg, following an incompatible RBC transfusion), RBC elution studies typically show reactivity for a specific non-ABO antigen. Note that elution studies are most useful for IgG-positive DATs. C3-positive DATs are frequently associated with IgM antibodies; such antibodies are poorly eluted from the RBC surface and, in most blood bank settings, few reagents exist to detect bound IgM.

Cold Autoantibody Screen, Thermal Amplitude, and Titers

Cold autoantibodies are classically IgM and may be difficult to detect by standard blood bank testing. Therefore, cold screens have been developed to help identify the presence/absence of a cold autoantibody as well as possible targets of the autoantibodies on the RBC surface. In a cold autoantibody screen, a patient’s plasma specimen (collected and maintained at 37°C) is subsequently mixed with adult screening cells (harboring the I antigen), cord-blood RBCs (harboring the i antigen), and the patient’s RBCs (to determine whether any reactivity against self is demonstrable); all testing is done at 4°C. In most forms of cold agglutinin disease (CAD) in adults, cold autoantibodies should show strong reactivity against self and RBCs possessing the I antigen. Such reactivity classically has been described in CAD associated with Mycoplasma pneumoniae infections. In contrast, cold autoantibodies showing strong reactivity against self and i-positive RBCs may suggest CAD associated with Epstein-Barr virus infection.

When clinical suspicion exists for CAD, the cold screen is only the first step because many patients can possess clinically insignificant cold autoantibodies. Differentiating a significant from insignificant cold agglutinin requires clinical correlation in addition to examining the thermal amplitude of the autoantibody and its titer. Thermal amplitude studies involve incubating the patient’s plasma with screening cells at temperatures ranging from 0°C to 4°C through 35°C to 37°C. Most clinically significant cold autoantibodies show reactivity (ie, hemagglutination) at temperatures greater than 27°C to 30°C. Regarding titers, several dilutions of patient plasma are mixed with screening cells at 4°C to determine the strength of the autoantibody. Although no formal guidelines exist, reports in the literature suggest that titers of greater than 1:64 to 1:256 are consistent with clinically significant CAD.

Donath-Landsteiner Test

Paroxysmal cold hemoglobinuria (PCH) is a rare cold AIHA. Most often encountered after infection or vaccination in children, and presenting with severe intravascular hemolysis, PCH is caused by an IgG autoantibody (referred to as biphasic hemolysin) targeting the high-incidence P antigen. The DAT is typically positive for complement only, because the cold-reactive IgG autoantibody dissociates from the patient’s RBCs at the warmer temperature at which the DAT is performed. Physiologically, the biphasic nature of PCH is manifested by autoantibodies binding to RBCs at cold temperatures and fixing complement (phase I). Then, as RBCs warm back toward 37°C, fixed complement is within its optimal thermal range and intravascular RBC hemolysis ensues (phase II). The Donath-Landsteiner test thus involves incubating the patient’s plasma with control RBCs at a very cold temperature (typically 0°C –4°C) allowing any cold-reactive IgG autoantibodies to bind and fix complement. This specimen is then incubated at 37°C, allowing fixed complement to lyse the RBCs. When a PCH autoantibody is present, there should be gross hemolysis observed in the plasma supernatant of specimens incubated at 0°C to 4°C followed by 37°C.

Summary for Complex Tests of Compatibility and/or Antibody Identification

The tests described earlier are used in the assessment of hematology/oncology patients who require transfusion care, or in whom an alloimmune or autoimmune process is suspected. A detailed description of the further diagnosis and treatment of AIHA is beyond the scope of this article and the reader is referred to the June 2015 edition of Hematology/Oncology Clinics of North America for in-depth reviews of warm and cold AIHA. Because of these recent reviews of AIHA, the remainder of this article focuses on the alloimmune response to blood group antigens in patients with hematologic/oncologic disorders.