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Review Article

Autoimmune Hemolytic Anemia in Children: Laboratory Investigation, Disease Associations, and Treatment Strategies

Blackall, Douglas MD, MPH*; Dolatshahi, Lily MD

Author Information
Journal of Pediatric Hematology/Oncology: April 2022 - Volume 44 - Issue 3 - p 71-78
doi: 10.1097/MPH.0000000000002438
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Abstract

The autoimmune hemolytic anemias of childhood represent an interesting spectrum of clinical conditions with unique laboratory features, disease associations, and treatment strategies. They are rare in comparison to other childhood diseases and are less commonly seen than autoimmune hemolytic processes in adults.1 Though rare, they offer a unique window into immune dysregulation that can occur as a primary entity or be associated with well-described clinical conditions.

In this review, the 3 major forms of autoimmune hemolytic anemia in children will be discussed: warm autoimmune hemolytic anemia (WAIHA), cold agglutinin disease (CAD), and paroxysmal cold hemoglobinuria (PCH). Although rare cases of drug-induced hemolytic anemia have a true autoimmune origin, most of these cases are characterized by a novel alloimmune response. As such, they will not be discussed in this review.

LABORATORY BACKGROUND

By their nature, autoimmune hemolytic anemias are characterized by a self-directed humoral immune response (ie, an antibody). The nature of the offending antibody (eg, immunoglobulin [Ig] G or IgM; warm-reactive or cold-reactive) in large part determines the patient’s clinical presentation and the treatment strategies that will be considered. Before turning to a more detailed discussion of the autoimmune hemolytic anemias of childhood, it is important to discuss the laboratory evaluation of these disorders as (1) the diagnosis of these autoimmune processes hinges on the laboratory identification of an autoantibody and (2) the in vitro reactive nature of the offending autoantibody largely defines the specific type of autoimmune hemolytic anemia experienced by the patient (ie, WAIHA, CAD, PCH).

The laboratory identification of a red cell-directed autoantibody almost always occurs in a hospital blood bank or an advanced immunohematology reference laboratory. The reason for this is that these laboratories have the serological tools and expertise necessary to detect and characterize these antibodies. There are 2 primary tests that are used to detect red blood cell (RBC) antibodies, whether autoantibodies or alloantibodies (ie, directed against discreet blood group antigens): the indirect antiglobulin test (IAT) and the direct antiglobulin test (DAT).2 It was more common in the past to refer to these tests as Coombs’ tests, but this eponym is used less commonly in favor of more specific immunologic terminology (ie, antiglobulin). Regardless, the IAT detects antibodies in the plasma, while the DAT detects antibodies (and complement) associated with the red cell membrane. Additional and specific features of antiglobulin testing will be addressed in more detail with the discussion of each of the autoimmune hemolytic disease processes. However, further elaboration on DAT testing is important.

As mentioned above, the DAT detects specific proteins bound to the surface of RBCs. The commercially available antiglobulin reagents are directed against IgG and the C3d component of complement.3 Antiglobulin reagents to other Ig classes (eg, IgA, IgM) are not commercially available but may be offered in specialized immunohematology reference labs. Regardless, anti-IgG and anti-C3d reagents detect these specific proteins on the red cell membrane, the binding of which may result in hemolysis (ie, the chief pathophysiological consequence of red cell autoantibodies).

The sensitivity of DAT testing is primarily determined by the reagents used in testing and the platforms used to detect red cell agglutination, the primary event that defines a positive immunologic reaction in blood bank serological testing. It is important to note that there is a “normal” number of IgG or C3d molecules attached to the red cell membrane (~50 molecules of each), but the sensitivity of commercially available antiglobulin reagents is significantly higher (250 to 500 molecules of each).3 This has 2 important clinical implications. First, a patient could have excess IgG or C3d on their red cells and might even experience active hemolysis, but the DAT could be negative as the amount of IgG/C3d is below the limit of detection. This is at least one explanation for the so-called “Coombs’-negative” hemolytic anemias.3 Second, patients can have positive DAT testing simply because they have excess IgG or C3d in their plasma. This results from a dynamic relationship between plasma concentrations of IgG and C3 and the number of these molecules that passively adsorb to the red cell membrane.1 The consequence of this is that patients who are hypergammaglobulinemic or hypercomplementemic may have positive DAT testing, usually weakly-reactive, but be at no downstream risk for hemolysis due to passive adsorption rather than an actively directed and specific autoimmune response.1 In fact, positive DAT testing is quite common among hospitalized patients for this reason, with ∼10% of hospitalized patients having positive DATs on surveillance testing (8.5% C3d only, 1.5% IgG only).1 In contrast, positive DAT testing is distinctly uncommon among healthy individuals, as evidenced by a positivity rate of 1 in 14,000 blood donors.1 In short, a positive DAT alone is insufficient to confirm the detection of an autoantibody, much less to make the diagnosis of autoimmune hemolytic anemia. At most, a positive DAT indicates a risk for hemolysis. See Table 1 for typical laboratory findings seen in the autoimmune hemolytic anemias of childhood.

TABLE 1 - Typical Findings in the Autoimmune Hemolytic Anemias
Type Etiology Ig Class Antibody Specificity Temperature of Maximum Reactivity DAT Primary Hemolysis
WAIHA Autoimmune/hematological conditions; may be primary IgG Usually Rh associated 37°C IgG±C3 Extravascular
CAD Lymphoproliferative disorder of bone marrow IgM Big I 4°C C3 Extravascular
CAS Secondary to infections and aggressive lymphomas IgM or IgG Big I or little i 4°C C3 Extravascular
PCH Secondary to infections IgG P 4°C binding; 37°C hemolysis C3 Intravascular
CAD indicates cold agglutinin disease; CAS, cold agglutinin syndrome; DAT, direct antiglobulin test; Ig, immunoglobulin; PCH, paroxysmal cold hemoglobinuria; WAIHA, warm autoimmune hemolytic anemia.

The laboratory diagnosis of an autoimmune hemolytic anemia begins with the identification of a RBC autoantibody through appropriate IAT and DAT testing. Once identified, however, additional laboratory testing is required to determine if the autoantibody is hemolytic in nature. Specifically, serial hemoglobin/hematocrit values are helpful along with the following laboratory tests and typical results: reticulocyte count (elevated), lactate dehydrogenase (elevated), haptoglobin (below the normal reference range or undetectable), and bilirubin testing (elevated total and indirect bilirubin).4 In addition, red cell morphology on the peripheral blood smear can sometimes be useful (eg, the presence of spherocytes in cases of WAIHA). Again, the detection of an autoantibody is insufficient to make the diagnosis of an autoimmune hemolytic process. The patient must also have laboratory evidence of hemolysis. Finally, in those patients who experience autoimmune hemolysis, the fate of the opsonized RBC is largely dependent on the molecule bound to the cell membrane: IgG-coated RBCs are removed from the circulation (ie, extravascular hemolysis) by reticuloendothelial cells in the spleen (primary) and bone marrow, while C3d-coated RBCs are primarily removed by Kupffer cells in the liver.3

AUTOIMMUNE HEMOLYTIC DISEASES OF CHILDHOOD

WAIHA

Warm-reactive autoantibodies are products of RBC-directed immune responses that are maximally reactive at 37°C.5 Typically, they react with a patient’s own RBCs but also react with all donor RBCs against which they are tested. This is to say that the patient’s DAT is positive, and the IAT is pan-reactive. However, in a minority of cases, the autoantibody has either a relative or an absolute specificity for a blood group antigen, most commonly a member of the Rh blood group system.5 Warm-reactive autoantibodies are usually polyclonal IgG antibodies, though IgA or monomeric IgM antibodies are sometimes seen.1 Complement (C3d component) may also be detected on the RBC membrane in association with these antibodies (Table 1).1 Most importantly, the clinical significance of a warm-reactive autoantibody is related to its ability to cause hemolysis (WAIHA). In addition, these antibodies are important because they can interfere with routine pretransfusion compatibility testing (ie, the detection of clinically significant blood group alloantibodies and the ability to provide cross-match–compatible units of blood).3

WAIHA is by far the most common type of autoimmune hemolytic process. In various series, ∼70% of all patients presenting with autoimmune hemolytic anemia had warm-reactive autoantibodies.1 WAIHA affects patients of all ages but, overall, is a rare diagnosis (∼1:75,000 population).1 Patients of all ages are affected, ranging from infants in the first few months of life to the elderly. However, there is a general increase in incidence throughout life with a dramatic rise occurring after the age of 50, with women being affected predominantly.1

The overall incidence of warm-reactive autoantibodies in pediatric patients is not known, but those associated with hemolysis likely occur in <1 in 100,000 children.1 It is unusual in the very young but has been documented to occur in utero.6,7 A review of autoimmune hemolytic anemia cases at the Mayo Clinic over a 20-year period (1994-2014), identified 35 pediatric patients.8 The median age was 10 years, and almost two third of the patients were male. Of note, only 3 of the patients were under 1 year of age, 2 of which had WAIHA. Thus, it is fair to say that WAIHA is unusual in infancy. Despite this, pediatric autoimmune hemolytic anemia is not a particular rarity in the children’s hospital setting. As an example, in a single institutional series spanning nearly 9 years, 42 pediatric patients with warm-reactive autoantibodies were identified in a hospital blood bank.5 Not all of these patients experienced hemolysis (ie, WAIHA), but the blood bank evaluations that these patients required, some multiple over time, represented about 17% of all antibody identification work performed in the hospital laboratory.

Warm-reactive autoantibodies may be idiopathic but are oftentimes associated with a variety of clinical conditions notable for immune dysregulation and a propensity for autoantibody formation (eg, hematological malignancies, autoimmune diseases, chronic infections, and inflammatory conditions).9 There also seems to be a relationship between the type of disease entity associated with warm-reactive autoantibody development and the propensity for hemolysis. As an example, in a large case series from University of California, Los Angeles (UCLA), only 29% of the patients (largely adults) with warm-reactive autoantibodies experienced hemolytic anemia.9 However, 69% of the patients with hemolysis had diseases classically associated with WAIHA: autoimmune disorders, including idiopathic thrombocytopenic purpura and hematological malignancies. Additional studies of adult patients with a specific disease entity, who also had warm autoantibodies, address this issue. In one study of patients with carcinoma and warm-reactive autoantibodies, 49 of a total of 99 patients experienced autoimmune hemolytic anemia.10 In a second study, 46 patients with the myelodysplastic syndrome had erythrocyte autoantibodies (warm, mixed warm and cold, or cold).11 Fifteen of these patients had autoimmune hemolysis, with 7 patients experiencing hemolysis secondary to warm autoantibodies alone.

The association between disease process, red cell autoantibody formation, and the propensity for hemolysis is also seen in pediatric populations. In one study of 42 patients with detectable warm-reactive autoantibodies, 57% of patients had evidence of WAIHA.5 These patients largely had autoimmune conditions and hematological disorders including leukemias and sickle cell disease. However, none of the 14 sickle cell patients in the study had laboratory or clinical evidence of excessive hemolysis. This is an important finding as warm-reactive autoantibody development is seen in both adult and pediatric sickle cell disease patients, particularly those who are alloimmunized.12 The infrequency of hemolysis in sickle cell disease patients with warm autoantibodies is further corroborated by a series of 14 pediatric patients, only 4 of whom experienced autoimmune hemolysis.13

As a final note, the strength of reactivity of DAT testing (1+ to 4+ agglutination scale) is a direct reflection of the amount of IgG and/or C3d associated with the red cell membrane and is predictive of hemolysis, both in adult and pediatric patients.5,9 In a largely adult study population, 83% of the patients with warm autoantibodies who experienced hemolysis had a DAT for IgG which was 2+ or greater in reaction strength.9 Sixty-three percent of patients with warm autoantibodies who did not experience hemolysis had a DAT for IgG which was 1+ or weaker in strength. In another study of 23 patients with WAIHA, 21 patients had a DAT that was 4+, and 2 patients had a DAT that was 3+.14 This relationship also holds for pediatric patients. In a series of 42 pediatric patients with warm-reactive autoantibodies, the DAT was 2+ or greater in reaction strength in 16 of 24 patients (67%) with WAIHA. The DAT was 1+ or weaker in strength in the 14 of 18 patients (78%) who did not experience hemolysis.5 Thus, the DAT strength can be an important determinant of hemolysis propensity. Unfortunately, in electronic health records, DAT results are typically only reported as positive or negative (ie, without laboratory reaction strengths). However, this information can usually be obtained through direct communication with the blood bank laboratory and may have implications for the extent of monitoring that patients should receive.

Treatment

Because of a paucity of randomized and controlled studies in pediatric patients with WAIHA, existing therapeutic recommendations are mostly based on retrospective studies, case reports, or treatment in adult populations.15 Treatment depends on the severity and rapidity of anemia development, the type of antibody involved (IgG vs. IgM), and whether the WAIHA is primary or secondary to an underlying disorder. Almost all patients will need admission to the hospital at the time of initial diagnosis.16 A first step in management is to determine if a patient needs to be transfused.

Transfusion

The decision to transfuse depends on the severity of anemia and the associated symptoms related to the anemia. As an example, an otherwise stable child with a hemoglobin level of 9 to 12 g/dL may require observation alone. If the patient is symptomatic, with lethargy or shortness of breath, or if the rate of hemoglobin decline is rapid, with hemoglobin between 6 and 9 g/dL, transfusion therapy may be indicated. For patients who have very severe anemia with hemoglobin levels <6 g/dL, RBC transfusion should be instituted as soon as possible.15,16 As mentioned above, the offending autoantibody in WAIHA is typically directed against blood group antigens that are present on all RBCs. Thus, these autoantibodies may interfere with routine pretransfusion antibody screening and compatibility testing. Therefore, finding truly matched RBC units is generally impossible, but transfusion can be safely performed if underlying alloantibodies are excluded. Ultimately, the physician must balance the risk of delaying transfusion against the benefit of RBC transfusion in a patient with severe anemia.17

Corticosteroids

First-line pharmacotherapy for WAIHA is corticosteroids, typically given as prednisone/prednisolone at a starting dose of 1 to 2 mg/kg/d for 1 to 3 weeks until the hemoglobin is >10 g/dL. Thereafter, a slow and gradual tapering over a period no shorter than 4 to 6 months is recommended. Overall, 81% to 100% of children with primary or secondary WAIHA respond to corticosteroids.18,19 In patients with particularly rapid hemolysis and very severe anemia, methylprednisolone, given at a dose of 1 to 2 mg/kg, is typically provided every 6 hours. Once the hemoglobin level begins to rise and the patient is clinically stable, oral prednisone should be used at a dose of 2 mg/kg/d for younger children and 1 mg/kg/d for adolescents. High doses of steroids are usually required for 2 to 4 weeks. Most importantly, the steroid taper must be performed slowly, over a period 3 to 6 months, to avoid relapse. When a patient relapses, a very high dose of prednisone is usually required to again achieve remission.16,19 Thus, the total duration of corticosteroid therapy is prolonged and can vary from 3 to 12 months after remission is achieved. In a French retrospective study of WAIHA, the overall clinical response to corticosteroids was 80%. After a median follow-up of 26.6 months, ∼20% of patients had relapsed.8 Ultimately, the goal is to maintain a stable hemoglobin value with a relatively low dose of corticosteroids. For a child with chronic or refractory WAIHA, more aggressive therapy is often required to maintain a stable hemoglobin and promote a high quality of life. Long-term use of corticosteroids is undesirable given their numerous adverse effects including hypertension, hyperglycemia, irritability, and psychosis. Finally, the maximum daily dose of prednisone is typically 60 to 80 mg.16

Rituximab

Rituximab and splenectomy are the only second-line treatments with proven short-term efficacy. However, because of the complications related to splenectomy, rituximab is usually considered to be the second-line therapy of choice. Rituximab is a monoclonal antibody specific for the CD20 antigen, which is expressed on B lymphocytes. It can cause rapid depletion of both normal B lymphocytes and lymphoma B cells. Patients who are taking corticosteroids before initiating rituximab should continue to take steroids until a response to rituximab is clearly established. In a meta-analysis of 154 patients with WAIHA, the overall response rate to rituximab was 79%, although not all cases were primary, and some received concomitant steroids.20 A Danish study randomized 64 patients to prednisolone only or prednisolone with rituximab (375 mg/m2 weekly for 4 wk). The response rate to prednisolone and rituximab was significantly higher at 12 months (75% vs. 36%) with no difference in adverse reactions.21 A French study randomized 32 patients to prednisone-placebo versus prednisone-rituximab (1 g on days 1 and 15). Again, the primary endpoint of overall response at 12 months was significantly higher in patients receiving rituximab (75% vs. 31%) without excess adverse events.22 Ideally, before considering the combination of steroids with rituximab as first-line therapy, larger studies of longer duration are needed. As a final note, rare complications of rituximab therapy include severe infusion reactions, infection, progressive multifocal leukoencephalopathy, and reactivation of hepatitis B.

Splenectomy

Approximately 70% of patients with primary WAIHA respond to splenectomy.17 There appears to be an ∼30% rate of relapse in the short term, but unlike splenectomy for immune thrombocytopenia, the long-term durability of remission is unknown. Given that childhood WAIHA is often self-limited, splenectomy should usually be considered a third-line treatment option. Because of the risk of postsplenectomy sepsis with encapsulated bacterial organisms, children should be immunized with the polyvalent polysaccharide vaccines against Streptococcus pneumoniae and Neisseria meningitidis before surgery. This is in addition to routine infant immunizations against S. pneumoniae and Haemophilus influenzae. After splenectomy, all children should receive twice-daily penicillin prophylaxis for at least 5 years after surgery.23 High-risk patients (eg, those with a history of sepsis or an underlying immune deficiency) require lifelong prophylaxis.24

Intravenous Immunoglobulin (IVIG)

IVIG has been used as a rescue therapy for those with severe transfusion-dependent hemolysis. Overall, approximately one third of patients will respond to IVIG. In one series, 6/11 (54.5%) children responded to doses of 2 to 4 g/kg/d for 2 to 5 days.25 As such, IVIG may be a useful rescue therapy.

Alternative Therapeutic Agents

Other agents have been used to treat refractory WAIHA, but published data on their efficacy are limited to case reports. A response to many of these agents may take months. As such, treatment with these agents generally should be continued for up to 6 months before it is considered to have failed.16 Overall, although still widely used in clinical practice, these immune-suppressive agents are now recommended as third-line therapy after more effective and less toxic therapies are exhausted.

High-dose cyclophosphamide, without stem cell rescue, for refractory WAIHA has induced complete and partial remissions. Cyclophosphamide is administered as a daily oral dose of 50 to 100 mg or as an intravenous monthly dose of 800 mg/m2 for 4 to 5 cycles. There is a reported efficacy of 60% to 70%, but few sustained responses have been seen. Side effects include myelosuppression, infections, secondary malignancy, and fertility problems with potential teratogenicity.26,27

Azathioprine is usually given as a steroid-sparing agent at 2 to 4 mg/kg for at least 1 to 3 months with a response rate of 60% to 70%.19 Cyclosporine has improved the course of WAIHA in a few patients with the steroid-refractory disease, but it is not routinely used because of the adverse effects of nephrotoxicity, hypertension, and hirsutism.28 The use of 6-mercaptopurine in pediatric patients with WAIHA was reported as part of a case series of autoimmune cytopenias. All 7 pediatric patients with refractory WAIHA responded.29 However, the time required to achieve a hematological response was not reported. The adverse effects of 6-mercaptopurine include leukopenia, thrombocytopenia, and elevated transaminase levels.29

Mycophenolate mofetil, also known as MMF or CellCept, inhibits lymphocyte proliferation and was assessed prospectively in a single-center study for adult patients with refractory autoimmune cytopenias. The study included 3 patients with refractory WAIHA. All patients responded and achieved a hemoglobin level >10 g/dL; they were transfusion independent after 4 to 6 months, and the drug was well tolerated. After this response was achieved, none had relapsed at a follow-up of 49, 40, and 13 months. Similarly, sirolimus, which increases T-regulatory cells and induces apoptosis in abnormal lymphocytes, has been used successfully in children to treat refractory primary WAIHA.30 The typical doses of MMF and sirolimus are 600 and 2 mg/m2, respectively. These agents are usually reserved for refractory cases or cases occurring secondary to an underlying immune disorder.30

Finally, hematopoietic stem cell transplantation (HSCT) in patients with autoimmune hemolytic anemia has been reported with limited success. In a study of 36 patients with severe refractory autoimmune cytopenias, 2 patients underwent allogeneic HSCT, and 5 patients underwent autologous HSCT.31 The 2 patients who underwent allogeneic HSCT achieved continuous remission. Of the 5 patients who underwent autologous HSCT, 1 died of treatment-related causes, 1 died of progressive disease, 2 had no response, and 1 had a transient response. A single case report also describes successful autologous HSCT in a child with refractory WAIHA.32 Despite significant advances in the management of high-dose chemotherapy and HSCT, this treatment option should be reserved for patients with the severe, life-threatening disease for whom all other therapies have failed.31,32

Cold Autoimmune Hemolytic Anemia

Cold autoimmune hemolytic anemia, sometimes generically referred to as CAD, in the second most common form of autoimmune hemolytic anemia both in children and adults.1 It is estimated that ∼25% of all autoimmune hemolytic anemia cases are attributable to a cold agglutinin, with an overall incidence of 1 in 300,000 individuals.1 However, CAD appears to be more common among the elderly. Interestingly, there is a bimodal distribution of cases. Namely, it is primarily seen in children and older adults.33 The reason for this is directly related to the disease processes with which it is typically associated. In children, CAD most commonly occurs with acute infections, especially Epstein-Barr virus and Mycoplasma pneumoniae. Other infectious agents have also been implicated including influenza, varicella, and measles, among many others.34 In adults, CAD is most commonly seen in association with lymphoproliferative disorders including Waldenström macroglobulinemia and a variety of lymphomas.35 As with WAIHA, however, CAD can occur as a primary process, without an obvious disease association.33

Cold autoimmune hemolytic processes are sometimes subdivided into CAD, defined as a clonal lymphoproliferative disorder of the bone marrow, and cold agglutinin syndrome (CAS), defined as a cold autoimmune hemolytic anemia that occurs secondary to another distinct clinical disease.35 These are useful designations with respect to the immunologic and pathophysiological drivers of these disease processes, though they are less important when the focus is more clinical in nature. For example, in contrast to the polyclonal nature of warm-reactive autoantibodies, the autoantibodies in CAD and CAS associated with lymphoproliferative disorders are almost always monoclonal immune responses, usually with an IgM kappa immunotype.35 In contrast, secondary cases (CAS), as with infection, are almost always polyclonal immune responses.35 Even so, these autoantibodies tend to have the same target antigen specificity: the I and i blood group antigens.33

The I (big I) and i (little i) blood group antigens are the 2 members of the Ii blood group antigen collection.36 They are oligosaccharide antigens defined by linked repeating lactose disaccharides. The i antigen is a linear structure, while the I antigen is branched and results from the activation of a specific glycosyltransferase enzyme in early childhood.36 As such, the i antigen is predominant on neonatal and cord RBCs, while the I antigen is found on adult RBCs.36 This has relevance with respect to the autoantibodies found in the cold agglutinin-mediated hemolytic anemias. The IgM kappa autoantibodies commonly associated with CAD are monoclonal and have specificity for the I antigen.35 In contrast, the polyclonal immune response associated with infections typically has a I specificity with M. pneumoniae and a i specificity with Epstein-Barr virus.33

Blood bank serological altesting is designed to maximize the detection of clinically significant antigens and antibodies and minimize the detection of immunological reactions that are irrelevant clinically.2 To that end, in vitro testing for blood group antibodies is carried out at 37°C to maximize the detection of clinically significant IgG blood group antibodies. This testing condition readily detects IgG warm-reactive autoantibodies but is less sensitive at detecting IgM cold agglutinins even though all RBCs routinely used for blood group antibody testing are from group O healthy adult donors with expected strong expression of the I antigen. As such, cold agglutinins may have variable reactivity with reagent RBCs, from no reactivity at all, to sporadic reactivity, to pan-reactivity.3 In large part, this is due to the thermal amplitude of the cold agglutinin, which is the maximal temperature at which a cold agglutinin will bind to its corresponding antigen.33

In typical cases of cold autoimmune hemolytic anemia, whether CAD or CAS, the DAT will be reactive for complement (C3d) and negative for IgG.3,33 Although cold agglutinins are almost invariably IgM antibodies, commercially available reagents for IgM red cell membrane detection (ie, anti-IgM) are not available. Thus, C3d serves as a surrogate marker for IgM that was once bound to the RBC membrane in vivo.3 Under typical circumstances, clinically significant IgM cold agglutinins are felt to bind to RBCs in the cooler parts of the body (eg, fingertips, toes, external ears) and activate complement to the C3 level. When RBCs reenter the warmer core circulation, the IgM cold agglutinin disassociates from the RBC membrane leaving C3 bound.33 Thus, the failure to detect IgM in routine DAT testing is a product of 2 issues: (1) lack of a commercially available reagent to detect IgM (ie, an IgM antiglobulin reagent) and (2) likelihood that any bound IgM might already have disassociated from the RBC membrane before in vitro DAT testing. This said, advanced immunohematology reference laboratories will usually be able to test for RBC-associated IgM due to research reagents that are available to them. However, in routine clinical practice this testing is not required; routine DAT testing for C3d is sufficient (Table 1).

There are 2 additional tests that are sometimes performed to assess the clinical significance of a cold agglutinin (via IAT and DAT testing): a cold agglutinin titer and thermal amplitude testing.33 The former is considered a routine part of serological testing when a clinically significant cold agglutinin is suspected, while the latter is typically reserved for less common cases in which the clinical significance of a cold agglutinin is unclear. The cold agglutinin titer reflects the overall strength of the antibody, which is primarily determined by concentration and avidity.33 The titer is determined by testing serial dilutions of serum or plasma against RBCs at a refrigerated temperature (1 to 6°C), as cold agglutinins will have greatest potency under these conditions. It is very common for healthy individuals to have detectable cold agglutinins when tested in this manner for unknown reasons, but when the titer exceeds 64 (the reciprocal of the last dilution for which reactivity is detected macroscopically), it is considered to be abnormal.35 However, most patients with hemolysis will prove to have significantly higher titers, with a median of 512 in several recent studies.33

A high-titer cold agglutinin is a general prerequisite for pathogenicity (ie, hemolysis), but it is insufficient. At least as important is the thermal amplitude of the antibody, which is the highest temperature at which the antibody will detectably bind antigen.33 Specifically, cold agglutinins of high titer are likely to be clinically significant with a thermal amplitude that exceeds ∼30°C.33 Those antibodies that fail to react at temperatures approximating this value are unlikely to be hemolytic, irrespective of titer. Likewise, those antibodies that are reactive at temperatures approximating core body temperature (37°C) are more likely to be clinically significant even though their titer may be lower.33 As mentioned above, the assessment of thermal amplitude is not a regular component of a cold agglutinin evaluation and would typically only be offered in higher level immunohematology reference laboratories. This is due to the fact that the testing procedure is more complicated, and it is usually not indicated to make the diagnosis of a cold autoimmune hemolytic process. In short, thermal amplitude testing is most useful in rarer clinical situations in which a patient is experiencing hemolysis, but it is unclear whether there is an immunologic component or if a detectable cold agglutinin is causative.33 As an example, occasional patients will have mixed cold-reactive and warm-reactive autoimmune hemolytic processes. In such cases, it is sometimes important to determine if a cold agglutinin is pathologic to make the most rational treatment decisions.

It is important to note that the detection of a cold agglutinin alone is insufficient to render a diagnosis of CAD. In parallel with warm-reactive autoantibody testing, the detection of a cold agglutinin and an accompanying positive DAT (for C3d) may put an individual at risk for hemolysis, but these findings are insufficient to make a clinical diagnosis of autoimmune hemolytic anemia. This designation requires the in vivo removal of C3-opsonized RBCs to a degree that is clinically significant (ie, results in anemia). That said, higher titer cold agglutinins, those with a higher thermal amplitude, and those capable of high-level complement deposition on the RBC membrane are more likely to be clinically significant.33

Finally, cold autoimmune hemolytic processes are generally self-limited when associated with an infectious process but may result in a chronic hemolytic state when associated with a lymphoproliferative disorder of the bone marrow.35 In the latter case, hemolysis can be exacerbated by infections or exposure to the cold.33,34 In fact, the incidence of cold autoimmune hemolytic processes is higher in areas of the world with colder ambient temperatures.33 When hemolysis occurs, it is generally extravascular in nature. C3-opsonized cells are primarily removed by the phagocytic Kupffer cells of the liver. In severe cases, however, intravascular hemolysis can occur when the complement cascade is no longer held in check at the C3 level by complement regulatory proteins (CD55 and CD59), and the cascade is taken to completion via the formation of the membrane attack complex.33 Though rare in CAD, this is a more common occurrence in cases of PCH.

Treatment

Because episodes of CAD can be transient, a period of support with transfusion may be useful before determining the need for pharmacological intervention. Patients with mild, asymptomatic anemia can be monitored without specific treatment. Nonpharmacological management consists of thermal protection: avoiding cold exposure and cold infusions. If transfusion is indicated, most literature recommends the use of an in-line blood warmer. Transfusion, when indicated, is considered safe. As opposed to WAIHA, finding compatible blood is generally not an issue.16,20,35

Corticosteroids are generally ineffective in the treatment of CAD. As such, rituximab is the first-line therapy for this condition. Two prospective, nonrandomized studies using rituximab (375 mg/m2 for 4 cycles at 1-wk intervals) found a partial response in ∼50% of the patients, but a complete response was rare. The median response duration was 11 months (range, 2 to 42 mo), and 6 of 10 retreated patients achieved a second response.37,38

In severe cases of cold, IgM-mediated AIHA, plasmapheresis can efficiently remove autoantibodies, and it may improve the clinical course of disease. Daily or alternate-day plasma exchange of 1 to 1.5 times the plasma volume with albumin replacement has been used as a bridging therapy, although evidence is largely confined to case reports.20

Patients with acute, severe intravascular hemolysis may also respond to the C5 inhibitor eculizumab, which blocks the terminal complement pathway. A randomized prospective trial that included 12 patients with CAD and 1 with severe CAS demonstrated some effect of therapy with eculizumab.39

Small retrospective series and case reports indicate that splenectomy does not have a role in the treatment of CAD. Therapy with corticosteroids and immunosuppression are generally ineffective as well.39 The response rate to corticosteroids was <20% in a retrospective series of 86 patients. However, the combination of fludarabine and rituximab was studied in a prospective, nonrandomized trial of 29 adult patients. This regimen (rituximab, 375 mg/m2 on day 1 and oral fludarabine, 40 mg/m2 on days 1 to 4 for 4 cycles at 28-d intervals) yielded an overall response rate of 76%. The Median estimated response duration was 66 months, but 14% of patients had grade 4 neutropenia, and 59% experienced grade 1 to 3 infections.40

As secondary CAS is even rarer than CAD, no systematic study has been published in this group of disorders. Recommendations have been based on case reports and expert opinion. In CAS associated with aggressive lymphoma and other malignancies, no therapy has been established except for treatment of the underlying disease.35 In infection-associated CAS, optimal antimicrobial therapy should be instituted. Whereas appropriate antibiotic therapy for M. pneumoniae is usually effective for control of the infection, the onset of secondary CAS will often occur after antibiotic therapy has been initiated or even completed.41

PCH

PCH is the least common of the autoimmune hemolytic anemias accounting for <5% of cases in most series.1 However, it appears to be more common in children than adults, which is related to the pathophysiology of the condition.34 Historically, PCH was primarily an adult disease that was sometimes seen in association with tertiary syphilis.34 However, it is now almost exclusively a disease of childhood associated with a wide variety of infectious agents including measles (and measles vaccination), mumps, cytomegalovirus, infectious mononucleosis, chickenpox, as well as M. pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, and Escherichia coli.42 Sudden, acute attacks of hemolysis typically occur during the time that a pediatric patient is recovering from a recent upper respiratory tract infection.34 Interestingly, the designation “PCH” is a misnomer when considering childhood cases of the disease as clinical manifestations are rarely paroxysmal, seldom precipitated by cold, and not necessarily characterized by hemoglobinuria, though this was a more common historical presentation in adults.42

PCH was first characterized by Donath and Landsteiner in 1904.43 The implicated autoantibody, often referred to as the Donath-Landsteiner antibody, is a polyclonal IgG agglutinin that has strong complement-fixing properties.42 From a serological standpoint, the autoantibody is described as a biphasic hemolysin.42 Both in vitro and in vivo, the autoantibody binds to RBCs at cooler temperatures and induces hemolysis at warmer temperatures that are more conducive to complement activation.43 Some red cells are coated with C3 and are cleared by the liver (ie, extravascular hemolysis), while other cells are lysed through activation of the terminal complement components (ie, intravascular hemolysis).43

In most cases, the Donath-Landsteiner autoantibody has specificity for the P blood group antigen, which is also the cell surface receptor for Parvovirus B19.42 The P antigen is a glycolipid component of the red cell membrane, but it is widely distributed on microorganism surfaces.43 The PCH causative autoantibody, therefore, may represent a cross-reacting immunologic response to antigens present on microorganisms.43 Since the pathogenic anti-P autoantibody is cold-reactive, it would not typically be detected in routine IAT testing, similar to cold agglutinins. Although these antibodies are IgG, they are rarely identified with anti-IgG reagents during DAT testing due to the disassociation of IgG from the RBC membrane at core body temperature.42 Instead, the DAT is typically strongly reactive for C3d in those who experience hemolysis (Table 1).42 As such, a high index of suspicion will likely be required to consider a PCH diagnosis. When suspected, however, the Donath-Landsteiner test may be indicated. This serological test is designed to mimic the biphasic nature of the causative autoantibody.33 Specifically, the patient’s serum is mixed with compatible donor RBCs at 4°C along with fresh normal serum as a source of complement. The mixture is incubated at 4°C and then warmed to 37°C. If the Donath-Landsteiner autoantibody is present, hemolysis will occur with warming.33 As can be imagined, this testing is complicated and is generally only available at immunohematology reference laboratories. In a typical clinical context, however, it may not even be needed as the hemolytic episode in most patients is self-limited, though it can be severe.34

Treatment

Supportive care is the mainstay of therapy for PCH due to the transient nature of the illness. In the acute phase, intravascular hemolysis can be severe, and blood transfusion may be required. Although the specificity of the Donath-Landsteiner antibody is typically the P blood group antigen, P antigen-negative blood is rare and is not usually required; most patients achieve an adequate response to transfusion with P-positive RBCs.44 Fever should be managed with antipyretics, but active cooling should be avoided due to the risk of precipitating hemolysis. Although cold avoidance has been recommended, there is no evidence to support the active warming of patients. Steroids are reserved for severe or persistent disease. In the setting of life-threatening disease, plasmapheresis may temporarily reduce the hemolytic burden.17

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Keywords:

anemia; autoantibodies; hemolysis

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