Introduction

Evans syndrome occurs when your body's immune system attacks and destroys your own blood cells. It attacks at least two of the different blood cell types, leaving you with a deficit of red blood cells (RBCs), platelets and/or white blood cells. Early and accurate diagnosis is important in order to provide treatment and reduce morbidity and mortality risk. The Direct Antiglobulin Test (DAT/Coombs test) is an important diagnostic tool that can aid in confirming Evans syndrome diagnosis and eliminating possible differential diagnoses.

Evans syndrome: Overview

Evans syndrome is defined as the concordant or sequential association of two or more cytopenias (low numbers of blood cells), commonly warm autoimmune haemolytic anaemia (AIHA) (when your immune system attacks and destroys your red blood cells leading to a shortage of red blood cells) with immune thrombocytopenia (ITP) (when the immune system attacks and destroys the platelets leading a platelet shortage), and occasionally with autoimmune neutropenia (autoimmune distruction of neutrophils, a type of white blood cell), which occurs in approximately 15% of cases.^1,2 

Evans syndrome is a rare situation representing around 7% of AIHA cases and 2% of ITP.¹ The mean age at diagnosis is 52, and as with other autoimmune conditions, the disease is more prevalent in females than males.² 

When AIHA and ITP occur concordantly, diagnostic tests are required to eliminate differential diagnoses and confirm Evans syndrome. Possible differential diagnoses include: thrombotic microangiopathies, anaemia following blood loss linked to bleeding problems complicating ITP, vitamin deficiencies, myelodysplastic syndromes, paroxysmal nocturnal haemoglobinuria, or specific conditions like HELLP when occurring during pregnancy. There are diagnostic challenges for confirming Evans syndrome due to overlap with other autoimmune cytopenias.²

The type of AIHA that occurs in Evans syndrome, warm AIHA, meaning that the IgG antibodies react with red blood cell surface antigens at body temperature. In ITP, the immune system is reacting against the GPIIb/IIIa antigen (marker) on the surface of platelets.²

Evans syndrome can be classified as primary (when the disease arises spontaneously) or secondary (when Evans syndrome develops as a result of another underlying condition). These classifications affect treatment options. Secondary Evans syndrome is linked with disorders such as lupus, common variable immunodeficiency, autoimmune lymphoproliferative syndrome, lymphoma, leukaemia and viral infections. Diagnosis of secondary Evans syndrome is important as the cytopenias tend to be more severe when associated with Evans syndrome than when presenting alone as AIHA or ITP.² 

While the exact cause of Evans syndrome is unknown, it is an autoimmune disorder in which your body's B cells produce ‘auto-antibodies’ which attack your own red blood cells, white blood cells and platelets.2 The pathology of Evans syndrome stems from the immune-mediated destruction of red blood cells, platelets, and sometimes neutrophils. Clinical features include anaemia, jaundice, fatigue, bleeding tendency and bruising. The specific signs and symptoms are variable and depend on the types of blood cell lines that are impacted. AIHA can present with fatigue, pallor, dizziness, shortness of breath, jaundice, and an enlarged spleen. With ITP, easy bleeding and bruising can occur, and with neutropenia, recurrent infections are common.²

For evaluating Evans syndrome, once anaemia is diagnosed via a complete blood count, further tests are needed to assess the levels of lactate dehydrogenase, haptoglobin and bilirubin and a reticulocyte count is needed to evaluate hemolysis. The presence of spherocytes on peripheral smear and a positive direct antiglobulin test (DAT/Coombs test) further confirms warm AIHA for an Evans syndrome diagnosis.²

Direct Antiglobulin Test (DAT/Coombs Test): Overview

The principle of the Coombs test is the detection of auto-antibodies bound to red blood cells.

Autoantibodies are antibodies that by mistake bind to your body's own cells and can lead to an immune response against these self-cells; therefore, they are a key marker of autoimmunity. 

Antibodies themselves are proteins produced by B cells that function in immune responses; they can bind ‘antigens’, markers on cells or viruses, and this binding can signal the immune system to act. Autoantibodies trigger immune responses against one's own cells, leading to autoimmune diseases. The antiglobulin (also called Coombs) test detects autoantibodies that bind to RBCs, leading to hemolysis (destruction of RBCs) in the process called autoimmune hemolytic anaemia (AIHA).³ 

There are two types of Coombs test: either direct antiglobulin testing (DAT) or indirect antiglobulin testing (IAT), both of which require a blood sample to be taken that is then analysed in a lab. 

DAT aims to detect antibodies bound and attached directly to the RBCs. 

DAT entails a process of washing a collected blood sample with saline to remove any antibodies not bound to the RBCs, which could affect the test result. Following washing, a reagent is added consisting of IgG (a type of antibody) and/or complement C3 (a complement system protein). DAT is typically used to diagnose blood-related conditions, such as AIHA.³

On the other hand, IAT looks for any unbound autoantibodies against RBCs that could be present in serum. Firstly, the serum is isolated, and native RBCs are removed from the sample. The isolated serum is then incubated with foreign RBCs with known antigens (surface proteins). Antiglobulin reagent is then added, and the presence of agglutination (the RBCs ‘sticking’ together) indicates a positive result for autoantibodies. IAT identifies circulating antibodies that could bind to RBCs and induce RBC hemolysis, though it is more frequently used for RBC phenotyping and is used as a screening test prior to blood transfusion to see how an individual will react to transfusion; IAT is also frequently used in prenatal testing.³ 

DAT is more regularly used for the diagnosis of AIHA. The Coombs test is necessary when autoimmunity to red blood cells is considered as part of the diagnosis, such as in Autoimmune hemolytic anaemia, which is the most recognised cause of a positive Coombs test result.³

Role of DAT in evans syndrome

Primary role and diagnostic value 

In Evans syndrome, a positive DAT result confirms the presence of autoantibodies on the surface of RBCs. This highlights that AIMH is occurring; thus, a positive DAT test result is a key diagnostic indicator in Evans syndrome. Therefore, DAT is useful in the initial diagnosis when Evans syndrome is suspected. DAT is a useful test for eliminating non-immune causes of hemolysis, such as drug-induced hemolytic anaemia and other cytopenias that could be possible differential diagnoses to Evans syndrome, as they mimic the symptoms. A negative DAT result would suggest an alternative diagnosis to Evans syndrome, as it suggests a non-autoimmune cause of hemolysis is occurring, as no autoantibodies are bound to the sampled RBCs. A positive DAT result alone is not fully diagnostic for Evans syndrome, but a positive result when in combination with thrombocytopenia and/or neutropenia strongly suggests (and has high diagnostic value for) Evans syndrome. DAT is useful for confirming AIMH; therefore, it is a useful test for guiding therapeutic decisions, such as focusing on prescribing immunosuppressant drugs and drugs like rituximab.^2,4

Patterns of DAT Positivity that can be identified upon testing a collected blood sample with different reagents can help characterise the specific type of immune response. Some patients may only have IgG antibody bound to RBCs (so are IgG positive only), others may be C3 positive only or be both IgG and C3 positive. Charterising the autoimmune response can help guide treatment decisions and understand the specific disease process in each patient. The IgG-positive-only pattern is more prevalent and suggests warm AIMH. Any C3 positivity suggests complement activation and may indicate a different pathology. Therefore, identifying DAT positivity patterns by using mono-specific reagents is useful for diagnosis.⁵

Limitations of DAT

While a key test for the diagnosis of Evans syndrome, DAT does have some limitations, these include: The possibility of false negatives (a negative result that should actually be positive) that can occur when autoantibody levels are too low to be detected by the test.
False positives (a positive result that should actually be negative) could occur if washing steps did not remove unbound antibodies. Lastly, that DAT alone is not diagnostic and must be interpreted with other clinical and lab findings.^3,4

Summary 

In summary, DAT is a key test used in the diagnosis of Evans syndrome. The DAT (Coombs) test provides important evidence of autoimmune hemolysis, essential for differentiating Evans syndrome from other cytopenias. However, while useful, DAT must be interpreted alongside clinical features such as thrombocytopenia and/or neutropenia and other investigations for accurate diagnosis and management.