Laboratory Evaluation of Immune Hemolytic Anemias

Etiology and Epidemiology

Etiology

Immune hemolytic anemia encompasses a spectrum of disorders characterized by the destruction of RBCs mediated by the immune system. The etiology can be categorized into autoimmune, alloimmune, and drug-induced mechanisms.

Autoimmune hemolytic anemia: AIHA occurs when the immune system produces autoantibodies that target self-antigens on RBCs, leading to their destruction. AIHA can be classified into warm AIHA, cold agglutinin disease, and mixed-type AIHA.

• Warm AIHA is the most common subtype, characterized by autoantibodies of the IgG class that react optimally at body temperature. However, IgA and warm-acting IgM have also been reported.
• Cold agglutinin disease involves cold-reactive IgM antibodies that agglutinate RBCs at low temperatures, causing intravascular hemolysis upon rewarming.
• Mixed-type AIHA exhibits features of both warm and cold antibody-mediated hemolysis.

Paroxysmal cold hemoglobinuria is an acquired hemolytic anemia characterized by an IgG autoantibody that activates complement at cold temperatures, leading to intravascular hemolysis upon rewarming, accompanied by hemoglobinuria (see Table 1. Common Secondary Conditions Associated With Various Autoimmune Hemolytic Anemias).[1]

Alloimmune hemolytic anemia: Alloimmune hemolytic anemia arises from the production of alloantibodies against foreign RBC antigens, often in the context of blood transfusions or hemolytic disease of the newborn. In hemolytic disease of the newborn, maternal antibodies directed against fetal RBC antigens lead to hemolysis in the fetus or newborn.  

Drug-induced immune hemolytic anemia: This anemia occurs when drugs or their metabolites produce antibodies that bind to and destroy RBCs. Frequently implicated drugs include penicillins, cephalosporins, and nonsteroidal anti-inflammatory drugs (NSAIDs). In addition, certain anticancer drugs, such as platinum compounds and immune checkpoint inhibitors, are common triggers.[2][3][4][5][6]

Table 1. Common Secondary Conditions Associated With Various Autoimmune Hemolytic Anemias

Epidemiology

The epidemiology of AIHA varies depending on the underlying etiology. Warm AIHA is the most prevalent subtype, with a bimodal age distribution peaking in young adulthood and later in life.[9] Warm AIHA shows a slight predominance in women.[3] Cold agglutinin disease predominantly affects older adults in their late 60s and early 70s and is often secondary to underlying conditions such as infections or lymphoproliferative disorders. Cold agglutinin disease is slightly more prevalent in women and accounts for 15% to 30% of all hemolytic anemia cases. 

Paroxysmal cold hemoglobinuria accounts for less than 1% of all hemolytic anemias. This condition is often considered a pediatric disease, with a median age of 5, and occurs in 57% of cases, especially in patients aged 10 or younger.[10] Mixed AIHA accounts for less than 10% of all hemolytic anemia cases. Alloimmune hemolytic anemia occurs in approximately 1 to 3 per 1000 pregnancies and is more common in individuals with a history of multiple transfusions. DIIHA is relatively rare but can occur in patients exposed to offending drugs.[11][12][13]

Pathophysiology

The detailed pathophysiology of autoimmune hemolytic anemias is mentioned below, focusing on the primary target antigens and corresponding DAT biomarkers (see Table 2. Pathophysiology of Autoimmune Hemolytic Anemia).

Warm Autoimmune Hemolytic Anemia

Warm AIHA occurs when the body's immune system mistakenly attacks its own RBCs, destroying them. This type of anemia can occur spontaneously without any apparent cause, known as primary or idiopathic warm AIHA, or it can develop secondary to certain underlying conditions or medications that trigger the production of autoantibodies targeting RBCs. The autoantibodies involved in warm AIHA are predominantly of the IgG type, although IgA and warm-acting IgM antibodies have also been reported. Subtypes of IgG, such as IgG1 and IgG3, can activate the complement system and cause more severe hemolysis than other subtypes. These antibodies typically target common antigens found on the surface of RBCs, with the most frequently targeted antigens including those of the Rh complex and glycophorin antigens, which are heavily glycosylated proteins on the membrane.[2][14]

In warm AIHA, the primary site of hemolysis is extravascular, predominantly within lymphoid organs such as the spleen. This process is primarily mediated by the Fc fragment of IgG through antibody-dependent cellular cytotoxicity. Minimal hemolysis is attributed to complement coating in warm AIHA. This leads to the formation of small, round cells called microspherocytes, which are less malleable than normal RBCs and can become trapped in the spleen's sinusoids, prolonging their destruction. In severe cases, intravascular hemolysis may occur if the reticuloendothelial system is overwhelmed or if the complement membrane attack complex is deposited on the surface of RBCs.[3]

Approximately 50% to 60% of warm AIHA cases are associated with underlying conditions, while the remaining cases are considered primary or idiopathic. Underlying conditions linked to secondary warm AIHA include various infections such as HIV, Epstein-Barr virus, hepatitis C, and, more recently, hepatitis E virus.[8] Recently, COVID-19-associated warm AIHA has been reported.[7] In addition, autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, or ulcerative colitis have been associated with warm AIHA.

Lymphoproliferative disorders such as autoimmune lymphoproliferative syndrome, chronic lymphocytic leukemia (CLL), lymphoma, and monoclonal gammopathies are also associated with warm AIHA. Immunodeficiency states, especially inherited immunodeficiency, hematopoietic stem cell transplantation, solid organ transplantation, and hypogammaglobulinemia, can also precipitate warm AIHA.[15] Rarely, pregnancy and medications can trigger warm AIHA. Some unique causes include Babesiosis in asplenic patients and bites from the Brown recluse spider.[16][17]

Cold Agglutinin Disease

Cold agglutinin disease is a rare autoimmune disorder characterized by autoantibodies, primarily of the IgM class, which exhibit reactivity against RBCs at temperatures below normal body temperature. Occasionally, IgG and IgA cold agglutinins have been reported, although IgA cold agglutinin does not cause cold agglutinin disease. The pathophysiology involves a complex interplay of immune mechanisms that culminate in the destruction of RBCs and subsequent anemia.[11][18][19] The underlying trigger for cold agglutinin disease is the production of autoantibodies by the immune system.[20] These autoantibodies typically target antigens, such as the I/i, GLOB, or P antigens, on the surface of RBCs.

When the body is exposed to colder temperatures, particularly below 37 °C, these IgM autoantibodies bind to multiple RBCs, causing agglutination or clumping—a process known as cold agglutination.[21] This phenomenon is the hallmark of cold agglutinin disease and is responsible for many clinical manifestations. In cold agglutinin disease, IgM autoantibodies to RBCs bind to C1q, triggering activation of the complement cascade, a critical part of the innate immune response.[22][23] Complement proteins, particularly C3b, are deposited on the surface of RBCs, which promotes their destruction. This complement activation enhances the opsonization of RBCs, making them more susceptible to phagocytosis by macrophages in the reticuloendothelial system, particularly in the liver by Kupffer cells, resulting in extravascular hemolysis.[24] Less commonly, in about 15% of cases, complement activation facilitates the formation of the membrane attack complex, leading to direct lysis of RBCs and subsequent hemoglobinuria, indicating intravascular hemolysis.[25]

Cold agglutinin disease is classified into primary (idiopathic) or secondary forms. Primary cold agglutinin disease involves cold agglutinin-mediated destruction of RBCs and extravascular hemolysis without an underlying disorder. Patients with primary cold agglutinin disease often have a low-grade clonal lymphoproliferative bone marrow disorder.[26] Secondary cold agglutinin syndrome is typically precipitated by an underlying infection, autoimmune disease, or overt lymphoma. In older patients, cold agglutinin disease often presents as a primary disease with an underlying lymphoid malignancy such as B-cell or plasma cell disorders, aggressive non-Hodgkin lymphoma, or Waldenström macroglobulinemia. Although solid tumors are rarely associated with primary cold agglutinin disease, this association is mostly uncommon.[27] Younger patients are more likely to have secondary cold agglutinin disease precipitated from infection, commonly M pneumoniae or Epstein-Barr infection.[28][29] Cases have been reported of infections precipitating secondary cold agglutinin disease from HIV, influenza, rubella, varicella-zoster virus, and COVID-19.[30][31][32][33][34]

Paroxysmal Cold Hemoglobinuria

Paroxysmal cold hemoglobinuria is an acquired hemolytic anemia characterized by an IgG autoantibody that activates complement in cold temperatures, resulting in intravascular hemolysis upon rewarming, accompanied by hemoglobinuria. The exact mechanisms triggering autoantibody formation remain unclear, but this typically arises in the context of infections or autoimmune disorders, suggesting immune stimulation of autoantibody production. A proposed mechanism involves the generation of cross-reacting antibodies that target viral or bacterial antigens mimicking the P antigen on RBCs.

The autoantibody in paroxysmal cold hemoglobinuria exhibits distinct characteristics, including specificity for the RBC GLOB antigen (formerly known as the P antigen)—a polysaccharide antigen on the RBC surface.[35] These antibodies are polyclonal, derived from multiple B-cell clones, and predominantly of the IgG class.[36] Unlike cold agglutinins, which are typically IgM antibodies that induce RBC agglutination, these antibodies do not cause agglutination but instead bind to RBCs below normal body temperature. Upon rewarming, the antibodies dissociate, but complement proteins remain attached to RBCs, leading to complement-mediated intravascular hemolysis—a hallmark of the anemia.

Diagnosis of the anemia relies on detecting the presence of the Donath-Landsteiner antibody, which fixes complement in cold temperatures, contributing to intravascular hemolysis upon rewarming.[37] Historically, paroxysmal cold hemoglobinuria was described as a chronic condition in patients afflicted with tertiary or congenital syphilis. However, currently, the anemia is often precipitated by infections, especially upper respiratory tract infections, gastroenteritis, and M pneumoniae. Autoimmune disease and lymphoproliferative disorders have been associated, although to a lesser extent (<10%) when compared to infectious etiology.[10]

Mixed Autoimmune Hemolytic Anemia

Mixed AIHA is a multifaceted hematological disorder characterized by the simultaneous presence of autoantibodies targeting different antigens on RBCs, resulting in hemolysis and anemia.[23] The immune pathology underlying mixed AIHA involves diverse mechanisms contributing to RBC destruction and associated clinical manifestations. Autoantibodies in mixed AIHA can target various RBC antigens, including Rh, Kell, Duffy, and others, reflecting the heterogeneous nature of the immune response in this condition.

The etiology of autoantibody production in mixed AIHA is often associated with underlying conditions such as autoimmune diseases, infections, or malignancies, which trigger immune dysregulation and stimulate the production of autoantibodies against RBC antigens.[21] This process may involve molecular mimicry, where antigens from infectious agents or tumor cells share structural similarities with RBC antigens, leading to the production of cross-reactive antibodies. Complement activation is a critical aspect of the immune pathology in mixed AIHA, as complement-fixing autoantibodies initiate the complement cascade, resulting in complement-mediated hemolysis.[22] This phenomenon involves depositing complement proteins on RBCs, which leads to their destruction through opsonization and the formation of the membrane attack complex.

Drug-Induced Immune Hemolytic Anemia

Immune-mediated destruction of RBCs primarily involves antibody-mediated mechanisms, where various antigen-antibody interactions lead to opsonization and subsequent phagocytosis by reticuloendothelial macrophages in the spleen and/or liver, resulting in extravascular hemolysis. While the DAT (or the direct Coombs test) is typically positive, occasionally, a drug metabolite rather than the parent drug itself may be responsible, complicating diagnosis. This anemia can be categorized by different mechanisms, including drug-dependent reactions and alteration of RBC surface antigens. In drug-dependent reactions, the drug binds to RBCs and becomes part of the antigen-antibody complex, remaining firmly bound to the membrane or causing immune complex formation. RBC surface antigens are altered when drugs modify normal membrane components, leading to immune hemolysis weeks to months after drug initiation.[38][39]

Oxidative stress from certain drugs can induce hemolysis, especially in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency or hemoglobin (Hb) H disease.[40] Oxidant injury forms oxidizing radicals, leading to hemolysis via oxygen radical damage to RBC membrane components and cellular proteins. Additionally, drug-induced hemolysis can result in methemoglobinemia and thrombotic microangiopathy, necessitating prompt recognition and management.[41] More than 130 drugs have been associated with DIIHA.[42] Immune hemolytic anemia is frequently triggered by antibiotics, particularly cephalosporins and penicillins. Certain anti-cancer drugs, such as fludarabine, platinum compounds, and immune checkpoint inhibitors, are common culprits.[5][6][43]

Table 2. Pathophysiology of Autoimmune Hemolytic Anemia

Alloimmune Hemolytic Anemia 

Alloimmune hemolytic anemia occurs when the immune system produces antibodies against RBC antigens perceived as foreign. This immune response can lead to the destruction of RBCs, resulting in anemia. Alloimmune hemolytic anemia can be triggered by several scenarios, most importantly incompatible blood transfusions and hemolytic disease of the fetus and newborn. Acute hemolytic transfusion reactions are typically caused by ABO incompatibility, most often due to clerical or procedural errors. These reactions involve the rapid destruction of RBCs when the recipient's immune system attacks transfused cells because they express an antigen foreign to the recipient. In rare instances, hemolysis can occur when transfused plasma contains antibodies targeting the recipient's RBC antigens. However, this is generally less severe due to the immediate dilution of donor antibodies upon transfusion.

The first step involves recipient antibodies attaching to antigens on transfused RBCs. This typically requires prior sensitization through previous exposure, such as a prior transfusion or pregnancy. Patients with blood groups O, A, and B continuously produce antibodies against the A and B antigens they lack, often due to exposure to similar antigens on intestinal microorganisms. These naturally occurring antibodies, particularly IgM, can quickly activate complement, leading to rapid intravascular hemolysis in cases of ABO incompatibility. In some cases, transfusion with blood products containing high titers of ABO alloantibodies can also trigger a reaction.

In hemolytic disease of the fetus and newborn, maternal sensitization in an RhD-negative individual occurs due to previous exposure to the Rh antigen, either through transfusion with Rh-positive RBCs or pregnancy with an Rh-positive offspring. Consequently, Rh hemolytic disease of the newborn generally does not occur in the first pregnancy in the absence of a transfusion history. Immunologically, antibody secretion begins with IgM, which cannot cross the placental barrier. This is later followed by isotype switching, leading to the production of IgG antibodies. IgG antibodies can cross the placental barrier, and they do so during the second and subsequent pregnancies, attacking the fetal RBCs and causing hemolysis and associated complications such as hydrops fetalis and jaundice.

Hemolytic transfusion reactions can involve both intravascular and extravascular RBC destruction. When an antigen-antibody complex forms, it may activate the classical complement pathway. Complete complement activation on the RBC surface results in the formation of the membrane attack complex and causes intravascular hemolysis. If complement activation halts at the level of C3b, RBCs coated with IgG and C3b are primarily destroyed by macrophages in the liver. Destruction of RBCs coated with C3b is less effective. RBCs coated only with IgG are removed from circulation in the spleen. These cells can also be destroyed by macrophages or through antibody-dependent cellular cytotoxicity involving large lymphocytes (K cells) releasing perforins. Both intravascular and extravascular hemolysis can occur in acute and delayed hemolytic transfusion reactions, although their relative contributions may vary.[13][44][45]

Diagnostic Tests

Diagnostic tests for the laboratory evaluation of immune hemolytic anemias are mentioned below (see Table 3. Serological Diagnoses of Autoimmune Hemolytic Anemia).

Complete Blood Count

CBC provides essential parameters such as hemoglobin concentration, hematocrit (Hct), RBC count, and mean corpuscular volume (MCV). In hemolytic anemia, a decreased hemoglobin and hematocrit occurs due to the loss of RBCs. Typically, hemoglobin in a patient with immune hemolytic anemia ranges from 7 to 10 g/dL, although hemoglobin less than 7 g/dL can be observed in up to 30% of patients. Common CBC findings associated with immune hemolytic anemia include reticulocytosis, normal or increased MCV, and an elevated reticulocyte percentage.[46] 

Haptoglobin

Haptoglobin binds free hemoglobin released from RBCs and is consumed during hemolysis. A decreased haptoglobin level indicates hemolysis, typically being low or undetectable in hemolytic anemia. A haptoglobin level of less than 25 mg/dL has an 87% probability of predicting hemolytic disease.[47]

Lactate Dehydrogenase

LDH is released from damaged RBCs and other cells during hemolysis. Elevated LDH levels are indicative of hemolysis, with median LDH levels around 500 units/L (U/L), although variations up to 5000 U/L have been reported. LDH levels are significantly elevated in intravascular hemolysis compared to only mild elevation in extravascular hemolysis.[20][48]

Reticulocyte Count

Reticulocytes are immature RBCs released from the bone marrow in response to anemia. An elevated reticulocyte count (>2.5%) suggests an appropriate bone marrow response to hemolysis. The absolute reticulocyte count is preferred, as it is unaffected by hemoglobin concentration. If the absolute reticulocyte count is unavailable, the reticulocyte percentage can be corrected for the hemoglobin. Reticulocytopenia can occur in AIHA, secondary to bone marrow infiltration by a lymphoproliferative disorder or to parvovirus B19 infection.[23][46]

Bilirubin

Increased levels of unconjugated bilirubin result from the breakdown of hemoglobin in hemolytic anemia. Indirect bilirubin is typically elevated in 2 to 3 mg/dL (35-51 μmol/L) in immune hemolytic anemia.[49]

Direct Antiglobulin Test or Direct Coombs Test

This test detects antibodies or complement proteins bound to the surface of patient RBCs, indicating immune-mediated destruction. A positive result confirms immune hemolysis.[50] This laboratory procedure involves the identification of antibodies or complement proteins bound to the surface of RBCs, indicating an immune response. The test is performed by collecting a blood sample from the patient, separating the RBCs from the plasma or serum, and washing the RBCs to remove any unbound antibodies or complement proteins. The RBC suspension is incubated with an antihuman globulin reagent, which specifically binds to antibodies or complement proteins attached to the RBC surface.

After incubation, the RBCs are rewashed to remove the unbound reagent, and the suspension is examined microscopically for signs of agglutination or antibody binding. A positive result, indicated by agglutination, suggests antibodies or complement proteins bound to the RBC surface, confirming the diagnosis of immune-mediated hemolysis.[40] The direct Coombs test may be performed in different variations, including tube and gel methods, but the basic principle remains the same. The severity of hemolysis is generally correlated with the strength of DAT positivity.

In patients with hemolytic anemia lacking recent transfusions or cold agglutinin or Donath-Landsteiner antibodies, a positive DAT for IgG and/or C3d is typically indicative of warm AIHA. Cold agglutinin disease is mainly characterized by DAT positivity for C3d (usually negative or weakly positive for IgG), indicating the involvement of IgM antibodies. For clinical significance, cold agglutinin titers are typically more than 512; however, levels above 64 are considered positive. Mixed AIHA manifests as DAT positivity for both IgG and high-titer cold agglutinins. Additionally, atypical forms of AIHA may occur, presenting as DAT-negative cases driven by IgA or warm IgM antibodies.[15] 

Paroxysmal cold hemoglobinuria is characterized by DAT positivity for C3d but negative for IgG/IgM. When evaluating alternatives, a positive DAT can also result from other conditions, including passive deposition of antibodies or immune complexes in liver disease, chronic infections, malignancies, systemic lupus erythematosus, renal disorders, and certain drug therapies such as intravenous immunoglobulin (IVIG) or anti-thymocyte globulin.[23]

Indirect Antiglobulin Test or Indirect Coombs Test

The indirect antiglobulin tests assess antibodies in the patient's serum that may react with donor RBCs. This test is essential for identifying alloantibodies and ensuring compatibility for blood transfusions.[51] This test is crucial for ensuring the compatibility of donor blood with the recipient's serum to prevent adverse reactions during blood transfusions and minimize the risk of hemolytic disease in the newborn. The procedure involves collecting a blood sample from the patient, isolating the serum containing antibodies, and incubating it with donor RBCs of known antigenic characteristics. 

After incubation, the RBC suspension is washed to remove unbound antibodies. An antihuman globulin reagent containing antibodies that bind to human immunoglobulins on the surface of RBCs is then added. Subsequent incubation allows the reagent to bind to any antibodies attached to the RBC surface. The RBC suspension is examined microscopically for agglutination or other signs of antibody binding, with agglutination indicating a positive result. This suggests that antibodies in the patient's serum react with the donor RBC antigens. This positive result confirms circulating antibodies targeting RBC antigens and supports the diagnosis of AIHA.

Furthermore, the indirect Coombs test aids in determining the specificity of the antibodies, which helps in guiding management decisions, including selecting appropriate blood products for transfusion and assessing the need for immunosuppressive therapy. Additionally, serial indirect Coombs testing can track changes in antibody titers over time, providing valuable insights into treatment response and disease progression in patients with AIHA.

Peripheral Smear

Examination of the peripheral blood smear allows visual inspection of RBC morphology, revealing characteristic changes such as spherocytes, schistocytes, and reticulocytosis indicative of hemolysis.[40] Spherocytes are commonly seen in warm AIHA and paroxysmal cold hemoglobinuria, whereas in cold agglutinin disease, spherocytes may be present but are not usually abundant. In DIIHA, the presence of bite cells, blister cells, or irregularly shaped RBCs (poikilocytosis) suggests oxidative hemolysis, while spherocytes or microspherocytes indicate immune hemolysis. Schistocytes, on the other hand, suggest a thrombotic microangiopathy such as disseminated intravascular coagulation.

Donath-Landsteiner Antibody

Donath-Landsteiner testing is reserved for patients with clinical signs of paroxysmal cold hemoglobinuria and a positive Coombs test for C3d. This assay identifies an antibody that adheres to RBCs in cold conditions, activates complement, and disassociates upon warming, a characteristic known as "biphasic hemolysin." However, this testing method can be time-consuming, resource-intensive, and prone to inaccuracies due to the low sensitivity.[52] Results may only show positivity for high-titer antibodies during a brief period around the hemolytic episode. Therefore, a negative outcome in a patient with a convincing clinical presentation cannot be considered evidence against diagnosing paroxysmal cold hemoglobinuria.

Osmotic Fragility Test

Osmotic fragility measures the ability of RBCs to withstand swelling and lysis in a hypotonic solution. Increased fragility is characteristic of certain types of hemolytic anemia, such as hereditary spherocytosis.

Autoimmune Serological Markers

Tests for autoimmune markers, such as antinuclear antibodies and rheumatoid factor, may assess underlying autoimmune conditions associated with immune hemolytic anemia.

Bone Marrow Aspiration or Biopsy

In some cases, especially primary cold agglutinin disease, a bone marrow sample may be obtained to evaluate for evidence of increased erythropoiesis, erythroid hyperplasia, or abnormal cell infiltration, which can provide diagnostic insights into the underlying cause of hemolysis.[4]

Additional Testing

Additional testing may include serum IgG and immunofixation if a cold autoantibody is suspected. Testing for HIV, hepatitis B, hepatitis C, and hepatitis E can help diagnose secondary causes of warm AIHA. If reticulocytopenia is present, testing for Parvovirus B19 IgM could be considered. Imaging studies may evaluate lymphoproliferative diseases. In cases of suspected DAT-negative AIHA, additional monospecific DAT for IgM, IgG, IgA, and C3d can be performed.[23][44]

Table 3. Serological Diagnoses of Autoimmune Hemolytic Anemia

Interfering Factors

Several factors can interfere with the laboratory evaluation of immune hemolytic anemia, affecting the accuracy of test results and diagnostic interpretation. Some of the key interfering factors are listed below.

Sample Collection and Handling

Improper blood sample collection or handling can lead to hemolysis, affecting test results. Collecting blood samples using appropriate techniques and anticoagulants prevents hemolysis during transportation and processing.[53]

Medications

Certain medications, such as corticosteroids, immunosuppressants, and blood transfusions, can interfere with serological tests for immune hemolytic anemia by affecting antibody levels or altering RBC antigen expression. Thus, the patient's medication history and the timing of drug administration should be considered when interpreting their test results.

Autoantibodies

Autoantibodies, such as cold agglutinins or warm-reactive autoantibodies, can complicate the interpretation of serological tests for immune hemolytic anemia. These autoantibodies may bind to RBCs nonspecifically, leading to false-positive results or masking the detection of specific alloantibodies.[40]

Transfusion History

Previous blood transfusions can result in donor-derived antibodies in the recipient's serum, leading to false-positive results on serological tests. Therefore, it is important to consider the patient's transfusion history and perform compatibility testing when interpreting test results.[54]

Underlying Medical Conditions

Underlying medical conditions, such as infections, autoimmune diseases, or malignancies, can affect a patient's immune system and antibody production, thereby influencing serological test results for immune hemolytic anemia. Evaluating the patient's clinical history and considering possible confounding factors when interpreting test results is important.[48]

Sample Quality

The reliability of test results can be compromised by factors affecting sample quality, such as sample contamination, inadequate volume, or improper storage conditions. Ensuring proper sample collection, labeling, and storage is essential to minimize the risk of sample-related interference.[55]

Technical Variability

Inconsistencies in test results can arise from variability in laboratory techniques, equipment, and reagents. Standardizing laboratory procedures and implementing robust quality control measures are essential to minimize technical variability and ensure the reliability of test results.[56]

Results, Reporting, and Critical Findings

The information below should be included in the laboratory report for hemolytic anemia.

• Patient information: Name, age, sex, and relevant medical history.
• Laboratory results: Results of CBC, peripheral blood smear interpretation, reticulocyte count, haptoglobin, LDH, bilirubin, Coombs test, and osmotic fragility test.
• Interpretation: Analysis of the results in the context of hemolytic anemia, including any findings to pinpoint the underlying cause.
• Recommendations: Further tests or consultations are recommended based on the findings, such as genetic testing for hereditary hemolytic disorders or additional imaging studies.

Critical findings in the laboratory evaluation of hemolytic anemia are mentioned below.

• Severe anemia: Hemoglobin levels below a critical threshold necessitate prompt intervention to avoid complications such as tissue hypoxia.
• High reticulocyte count: Indicates active bone marrow response to hemolysis, suggesting RBC destruction.
• Significantly elevated LDH: Suggests extensive hemolysis and tissue damage, requiring immediate investigation and management.
• Positive direct Coombs test: Indicates immune-mediated hemolysis, which may require specific treatment modalities such as immunosuppressive therapy.
• Characteristic morphological changes on peripheral blood smear: Presence of schistocytes, spherocytes, or other abnormal RBC morphologies indicative of specific hemolytic disorders.

Clinical Significance

Diagnosing the specific type of hemolytic anemia is crucial for tailoring effective management strategies and optimizing patient outcomes. Distinguishing between different etiologies, such as AIHA, alloimmune hemolytic anemia, and other forms of hemolysis, guides treatment decisions and interventions. Identifying whether the condition involves warm or cold antibody-mediated mechanisms in AIHA influences treatment selection. Warm AIHA, characterized by IgG-mediated hemolysis, typically responds well to initial treatment with glucocorticoids and rituximab, particularly in symptomatic patients.[3] Conversely, cold AIHA, involving IgM antibodies and complement activation, may necessitate different therapeutic approaches, such as avoidance of cold exposure and, in severe cases, plasmapheresis or immunosuppressive therapy. 

In symptomatic patients with cold agglutinin disease, whether or not a low-grade bone marrow lymphoproliferative disorder is detected, first-line treatment typically involves immunosuppressive therapy with rituximab monotherapy or a rituximab-bendamustine combination.[57] Paroxysmal cold hemoglobinuria is managed similarly to cold AIHA to avoid cold exposure; however, the efficacy of glucocorticoids has not been established.[10] The cornerstone of treatment in patients with DIIHA involves discontinuation of the offending medication. Symptomatic cases may benefit from glucocorticoid therapy.[58][59] Severe and relapsing cases may require further therapies such as IVIG therapeutic plasma exchange and/or rituximab. 

Diagnosing alloimmune hemolytic anemia, particularly in the context of hemolytic disease of the newborn or hemolytic transfusion reactions, informs immediate management strategies. Identifying maternal antibodies targeting fetal RBCs, typically anti-D antibodies, allows for timely interventions, including intrauterine transfusions or postnatal treatments to mitigate complications such as hydrops fetalis and neonatal jaundice.[60] Similarly, recognizing the presence of alloantibodies in hemolytic transfusion reactions prompts immediate cessation of transfusion and supportive care measures to prevent further hemolysis and life-threatening complications.

Enhancing Healthcare Team Outcomes

Utilizing a multidisciplinary approach significantly enhances healthcare outcomes in managing immune hemolytic anemia by capitalizing on collaborative efforts tailored to this complex condition. Timely diagnosis and treatment are imperative due to the potential for rapid disease progression and life-threatening complications. Unlike other forms of anemia, immune hemolytic anemia involves the immune-mediated destruction of RBCs, leading to acute anemia and associated symptoms such as fatigue, weakness, shortness of breath, and pallor.

Prompt diagnosis is essential to prevent severe complications, including hemolytic crises, acute kidney injury, cardiovascular instability, and death. The underlying autoimmune processes may also be associated with systemic manifestations or comorbidities that require timely identification and intervention. Given the urgency of prompt intervention, a multidisciplinary approach involving physicians, hematologists, immunologists, laboratory technicians, nursing staff, and pharmacists is crucial.

In this collaborative framework, the interprofessional healthcare team collaborates with hematologists and immunologists to interpret laboratory findings related to immune hemolytic anemia, diagnose underlying autoimmune processes, and devise individualized treatment plans tailored to the specific subtype of immune hemolytic anemia. Hematologists and immunologists contribute their expertise in understanding the pathophysiology and guiding treatment decisions. Laboratory technicians play a crucial role by performing the direct Coombs test, which detects immune-mediated hemolysis by identifying antibodies bound to RBCs. Their meticulous work ensures accurate and timely results, providing essential information for diagnosis and treatment monitoring.

Nursing staff are pivotal in patient advocacy, monitoring for signs of hemolysis, administering treatments, and educating patients and families about the disease process and lifestyle modifications. By harnessing the collective skills and expertise of the multidisciplinary healthcare team, prompt diagnosis and treatment are facilitated, thereby reducing the risk of complications associated with untreated immune hemolytic anemia. Effective coordination and communication among healthcare team members ensures comprehensive care delivery, ultimately improving healthcare outcomes and enhancing the quality of life for patients with immune hemolytic anemia.