Autoimmune haemolytic anaemia is a rare blood disorder where the immune system mistakenly attacks and destroys the body’s own red blood cells. Early and accurate diagnosis is essential for proper management and preventing serious complications, though the process requires a careful combination of clinical observation and specialized laboratory testing.

Introduction: Who Should Undergo Diagnostics

If you experience symptoms such as unusual tiredness, weakness, pale skin, yellowing of the skin or eyes, or dark-colored urine, it is advisable to seek medical attention. These warning signs may point to anaemia or red blood cell destruction happening in your body.^[1] Autoimmune haemolytic anaemia can develop gradually over several weeks, or in some cases, symptoms may appear within just a few days.^[1]

People with existing autoimmune diseases such as lupus, rheumatoid arthritis, or thyroid disease should be particularly alert to these symptoms, as they are at higher risk of developing secondary autoimmune haemolytic anaemia.^[1] Similarly, those with blood cancers like lymphoma or chronic lymphocytic leukaemia need to monitor for signs of anaemia, since the condition can arise as a complication of these illnesses.^[2]

Some people may not experience any symptoms at all, especially when the destruction of red blood cells happens slowly and the body has time to adjust. However, others may face severe symptoms that require urgent medical intervention.^[5] When symptoms are severe or develop rapidly, such as chest pain, shortness of breath, fainting, or symptoms of heart failure, immediate medical attention becomes critical, as untreated autoimmune haemolytic anaemia can be life-threatening.^[1]

Children can also develop autoimmune haemolytic anaemia, though it is very rare in this age group. In children, the condition is sometimes associated with other immune disorders, which makes prompt diagnosis even more important.^[3]

Diagnostic Methods: Identifying the Disease

Diagnosing autoimmune haemolytic anaemia involves a stepwise approach that begins with recognizing the presence of anaemia and then determining whether the cause is immune-related. Doctors use a combination of clinical assessment and laboratory tests to reach an accurate diagnosis.^[4]

Initial Blood Tests

The diagnostic process typically starts with basic blood tests that alert doctors to the possibility of haemolysis—the breakdown of red blood cells. A complete blood count will reveal a low number of red blood cells and haemoglobin, indicating anaemia. The anaemia is usually normocytic or macrocytic, meaning the red blood cells are normal-sized or larger than normal.^[4]

A blood test showing an elevated reticulocyte count is an important clue. Reticulocytes are immature red blood cells, and their increased number suggests that the bone marrow is working hard to replace red blood cells that are being destroyed faster than normal.^[4] However, in some patients, particularly at the time of initial presentation, the reticulocyte count may actually be low despite ongoing haemolysis. This can occur when the bone marrow is affected by an underlying condition or when the immune system attacks developing red blood cells.^[4]

Doctors also look for biochemical markers of red blood cell destruction. An elevated level of lactate dehydrogenase (LDH), reduced haptoglobin (a protein that binds free haemoglobin), and raised unconjugated bilirubin all indicate that red blood cells are breaking down.^[4] Haptoglobin levels decrease because the protein becomes depleted as it binds to haemoglobin released from destroyed red blood cells. Bilirubin, a yellow pigment formed when haemoglobin breaks down, accumulates and causes jaundice.^[2]

Blood Smear Examination

A blood smear, where a drop of blood is spread on a slide and examined under a microscope, can provide valuable diagnostic information. In autoimmune haemolytic anaemia, the smear may show spherocytes—small, round red blood cells that have lost their normal disc shape—or polychromasia, which reflects the presence of young red blood cells.^[4] In cold antibody types of the disease, red blood cells may clump together, a phenomenon called agglutination.^[4]

Direct Antiglobulin Test (Coombs Test)

The key test for confirming autoimmune haemolytic anaemia is the direct antiglobulin test, also known as the direct Coombs test. This test detects antibodies or complement proteins attached to the surface of red blood cells, confirming that the immune system is attacking these cells.^[4]

In warm autoimmune haemolytic anaemia, the most common type, the test is typically positive with anti-IgG antibodies. In some cases, it may also be positive for a complement protein called C3d.^[4] In cold autoimmune haemolytic anaemia, the test is usually positive for C3d because the cold-reacting IgM antibodies often detach from red blood cells at body temperature, leaving only complement behind.^[4]

It is important to note that the direct antiglobulin test can sometimes produce false-negative results. This may happen if the antibodies are of a type not detected by routine testing (such as IgA antibodies), if the antibodies are present in very small amounts, or if they bind weakly to red blood cells.^[4] Special testing techniques, including more sensitive methods like flow cytometry or solid-phase assays, may be needed in these cases.^[10]

Approximately 10% of people with autoimmune haemolytic anaemia have a negative direct antiglobulin test despite having the disease. In such cases, the diagnosis is made by excluding other causes of haemolysis and observing how the patient responds to treatment.^[10]

Distinguishing Between Types of Autoimmune Haemolytic Anaemia

Once autoimmune haemolytic anaemia is confirmed, doctors work to identify which type is present. The two main types are warm antibody haemolytic anaemia and cold antibody haemolytic anaemia, classified according to the temperature at which the antibodies are most active.^[1]

Warm autoimmune haemolytic anaemia involves IgG antibodies that bind red blood cells at normal body temperature. Symptoms usually develop gradually over weeks, though sometimes they appear within days. This is the most common form, accounting for the majority of cases.^[1]

Cold autoimmune haemolytic anaemia involves IgM antibodies that become active at temperatures below normal body temperature. This type affects only 10% to 20% of cases. People with this form may notice that their hands and feet turn blue or gray in color, and they may experience pain in the extremities when exposed to cold.^[1]

Identifying Underlying Causes

After diagnosing autoimmune haemolytic anaemia, doctors must determine whether it is primary (occurring without an obvious cause) or secondary (linked to another condition). In approximately half of cases, no underlying cause can be identified, and the condition is considered primary or idiopathic.^[2]

Secondary autoimmune haemolytic anaemia can result from other autoimmune diseases such as lupus, rheumatoid arthritis, or ulcerative colitis. It can also be caused by blood cancers such as lymphoma or chronic lymphocytic leukaemia, infections including Epstein-Barr virus, hepatitis, or HIV, or certain medications such as penicillin or some antibiotics.^[1][2]

To search for secondary causes, doctors may order additional tests such as imaging studies to look for signs of cancer, blood tests to check for autoimmune markers, or tests to identify recent or ongoing infections.^[4]

Distinguishing from Other Conditions

Because symptoms of anaemia can be caused by many different conditions, doctors must rule out other possible causes before confirming autoimmune haemolytic anaemia. This includes checking for inherited blood disorders, nutritional deficiencies, bone marrow problems, or other causes of red blood cell destruction.^[5]

Tests to exclude other diagnoses may include iron studies, vitamin B12 and folate levels, kidney and liver function tests, and in some cases, a bone marrow biopsy to examine how well the marrow is producing blood cells.^[4]

Diagnostics for Clinical Trial Qualification

When patients with autoimmune haemolytic anaemia are being considered for enrollment in clinical trials, additional diagnostic tests and specific criteria are typically required to ensure that participants are appropriate for the study and that results can be accurately interpreted.

Clinical trials generally require confirmation of the diagnosis through a positive direct antiglobulin test, along with documented evidence of haemolysis such as elevated LDH, reduced haptoglobin, or increased bilirubin levels.^[4] Trials may also specify minimum or maximum haemoglobin levels to ensure that participants have a certain degree of anaemia severity.

Studies investigating treatments for warm autoimmune haemolytic anaemia may require patients to have failed or relapsed after standard first-line treatments such as corticosteroids. Documentation of previous treatments and responses is typically needed.^[13] For cold autoimmune haemolytic anaemia trials, specific criteria related to the presence of cold agglutinins and their activity at particular temperatures may be required.

Additional laboratory tests may be needed to assess overall health and organ function before trial entry. These can include complete blood counts, comprehensive metabolic panels to check kidney and liver function, and tests to rule out infections or other conditions that might interfere with the study drug or affect safety.^[4]

Some clinical trials, particularly those testing new complement inhibitors or immunosuppressive agents, may require specific tests to evaluate immune system function or complement activity. Genetic testing or bone marrow examination might also be required in certain studies to better characterize the patient population or exclude other diagnoses.

Imaging studies such as chest X-rays or CT scans may be needed to screen for underlying conditions such as lymphoma or other cancers that could be causing secondary autoimmune haemolytic anaemia. Trials may exclude patients with certain secondary causes or may specifically recruit them, depending on the study objectives.^[4]

Throughout a clinical trial, regular monitoring is typically required to assess treatment response and safety. This includes repeated blood tests to measure haemoglobin levels, reticulocyte counts, markers of haemolysis, and the direct antiglobulin test. Frequency of testing is determined by the study protocol and may be more intensive than in routine clinical care.