24th May 2023

Haemolysis

Haemolytic Screen

Investigating haemolysis

Red blood cells have a lifespan of 120 days in the circulating bloodstream and are normally removed by macrophages in the spleen and liver. Premature destruction of red blood cells is termed haemolysis and leads to anaemia. There are several causes of haemolysis which can be classified based on the mechanism of red blood cell destruction.

A haemolytic screen is conducted when haemolysis is the suspected cause of anaemia and can help determine the underlying cause of haemolysis.

• Full blood count (including reticulocyte count)
  • Raised reticulocyte counts are seen in haemolysis as the body attempts to produce more red blood cells
• Peripheral blood film – to examine red blood cell morphology
• Liver function tests (including conjugated and unconjugated bilirubin i.e. “split bilirubin level”)
  • Hepatocytes are saturated in haemolysis and struggle to conjugate bilirubin, causing accumulation of unconjugated bilirubin in serum plasma
• Haptoglobin – a liver-produced protein that binds to free haemoglobin
  • Low levels in haemolysis
• Lactate dehydrogenase
  • Raised in haemolysis
• Direct antiglobulin test (DAT)

Direct Antiglobulin Test (Coomb’s Test)

This immunology laboratory test is used to determine if the cause of haemolysis is immune-mediated. Generally, immune haemolysis is due to antibody-mediated destruction of red blood cells. Antibodies that are directly attached to red blood cells result in a positive DAT. These antibodies can be auto-antibodies, as seen in autoimmune haemolytic anaemias, or, alloantibodies, seen in haemolytic transfusion reactions.

Inherited Haemolytic Anaemias

Membranous abnormalities

Hereditary Spherocytosis

Autosomal dominant disorder commonly found in Northern European individuals that affects the vertical linkage between the red blood cell membrane and cytoskeleton. This causes red blood cells (RBC) to lose their elastic deformability and results in the spherocytic shape of the RBC, instead of their characteristic bi-concave shape. The spherocytic shape of the RBC makes it more prone to haemolysis.

Clinical features – include jaundice, splenomegaly, megaloblastic anaemia, or may present as aplastic crisis secondary to Parvovirus B19.

Blood film – spherocytes, Howell-Jowell bodies, polychromasia

Treatment – supportive therapy, including blood transfusions as required, and splenectomy.

Haemoglobin abnormalities

Sickle cell disease and thalassaemia are inherited haemoglobinopathies that cause haemolysis and anaemia. These conditions are covered in the ‘Haemoglobinopathies’ section.

Metabolic abnormalities

Glucose-6-phosphate dehydrogenase (G6PD) deficiency

G6PD deficiency is an X-linked recessive inborn error of metabolism. G6PD is required in the metabolic pathway that causes reduction of NADP to NADPH, which is an important cofactor in glutathione metabolism. The main function of this metabolic pathway is to protect RBCs from oxidative stress and injury. Severity of haemolytic anaemia in G6PD deficiency depends on the variant of G6PD that is inherited. It can present as neonatal jaundice in newborns and cause kernicterus.

Clinical features – jaundice, and acute haemolysis precipitated by the following:

• Acute illness
• Drugs (a more comprehensive list can be found on www.g6pd.org)
  • Primaquine, fluoroquinolones, sulfonylureas, chloramphenicol, nitrofurantoin, rasbirucase, isoniazid
• Foods
  • Fava beans, henna compounds

Blood film – Microspherocytes, eccentrocytes (bite cells), blister cells, Heinz bodies, reticulocytes

Treatment – Avoidance of triggering drugs and foods, supportive management in haemolytic crisis

Pyruvate kinase deficiency (PKD)

PKD is a rare, autosomal recessive, inherited red blood cell enzyme disorder resulting in chronic haemolysis. Pyruvate kinase is a key enzyme in the glycolysis pathway. It produces ATP by converting phosphoenolpyruvate to pyruvate. It also modulates NADH production to reduce methaemoglobin levels. In PKD, reduced ATP production decreases RBC deformability (i.e. ability to change shape under applied stress without haemolysing), resulting in RBC damage and mainly extravascular haemolysis.

Clinical features – Neonatal jaundice, anaemia, hepatosplenomegaly secondary to haemolysis, frontal bossing due to extramedullary haemopoiesis

Blood film – normochromic cells, polychromasia, echinocytes (burr cells)

Treatment – Supportive therapy

Acquired Haemolytic Anaemia

Immune-mediated haemolytic anaemia

ABO and Rhesus Incompatibility

Typically, this is due to an immune response from the host to foreign allogenic RBC antigens from donor blood. It can also be less commonly due to plasma containing antibodies in donor blood that act against host RBC antigens.

Individuals with group A, B and O blood types develop IgM antibodies that react with the antigens they lack due to molecular mimicry following exposure to intestinal microorganisms that are similar to type A and type B antigens.

There are antibodies to other antigens on RBC (e.g. Kidd, Kell, Duffy) that can be present due to previous transfusions, pregnancy, or intravenous needle sharing. The presence of these can cause acute or delayed haemolytic transfusion reactions (HTR) depending on the titre of antibodies present. Acute HTR occurs during the transfusion, whereas delayed HTR can happen one week or more following the transfusion.

Both intravascular and/or extravascular haemolysis can occur in ABO incompatibility. Intravascular haemolysis is due to activation of complement when an antibody binds to the RBC antigen. Extravascular haemolysis occurs when the antibody opsonises the RBC causing phagocytosis in the reticuloendothelial system (namely, the liver and spleen).

Rhesus antigens are non-glycosylated transmembrane proteins in the RBC membrane. They are inherited independently of the ABO alleles. Rhesus incompatibility can cause haemolysis and Rhesus phenotyping is particularly important in pre-transfusion testing and prenatal care. Preventing alloimmunisation in Rhesus negative women of childbearing age is particularly important to reduce the risk of haemolytic disease of the newborn in a Rhesus positive pregnancy.

Warm Autoimmune Haemolytic Anaemia (AIHA)

Haemolytic anaemia due to autoantibodies that are active at body temperature is termed warm AIHA. It is the most common type of AIHA and can be spontaneous, or secondary to medication and illness.

Primary warm AIHA occurs with no precipitating or underlying condition.

Secondary warm AIHA can be associated with the following:

• Infection
  • HIV, CMV, VZV, Hepatitis C, pneumococcal infection, leishmaniasis, tuberculosis
• Autoimmune conditions
  • SLE, Sjogren’s, scleroderma, ulcerative colitis, sarcoidosis
• Immunological conditions
  • Combined variable immunodeficiency
• Lymphoproliferative disorders
• Pregnancy

Clinical features – jaundice, fatigue, pallor, shortness of breath, splenomegaly

Laboratory bloods – low haemoglobin, raised reticulocytes, low haptoglobin, raised lactate dehydrogenase

Blood film – spherocytes, reticulocytes

Direct Antiglobulin Test – positive, typically anti-Ig and/or anti-C3 (Note – Indirect antiglobulin test is not used to evaluate warm AIHA)

Management – transfusion in the acute setting, intravenous immunoglobulins or plasma exchange in critical situations, prednisolone and/or rituximab, azathioprine can be used as 3rd line, splenectomy in refractory cases of anaemia not responding to immunosuppression.

Cold Agglutinin Disease (CAD)

This is an autoimmune haemolytic anaemia that causes agglutination and extravascular haemolysis of RBCs at below core body temperatures. Cold agglutinins are typically IgM and recognise antigens on RBC in colder temperatures. Much like warm AIHA, it can be primary i.e. idiopathic, or secondary.

The causes of secondary CAD vary depending on the age of patients. Younger patients tend towards autoimmune and infective causes, while older patients may have underlying malignancy.

• Infection – Mycoplasma pneumoniae, Epstein-Barr virus
• Malignancy/Lymphoid disorders – lymphoma, Waldenstrom’s macroglobulinaemia, chronic lymphocytic leukaemia, monoclonal gammopathy of undetermined significance

Clinical presentation – Raynaud’s phenomenon, cold-induced acrocyanosis, symptoms of anaemia and haemolysis, splenomegaly, livedo reticularis, cutaneous ulceration or necrosis is severe cases

Laboratory bloods – As seen with other AIHA if significant haemolysis

Blood film – RBC agglutination

Red blood cell agglutination.

Image Source: Wikimedia Commons (2014). Red blood cell agglutination, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Red_cell_agglutination.jpg (Accessed on: 10 January 2023).

Direct Antiglobulin Test – Positive for C3, usually negative for immunoglobulin

Cold agglutinin titre – measured in suspected CAD, > 64 at 4 degrees Celsius

Management – Cold avoidance, treatment of underlying condition, blood transfusion in severe and symptomatic anaemia, plasmapheresis and intravenous IG as a temporary measure in acute haemolytic crises, rituximab for transfusion dependent/symptomatic anaemia/severe circulatory symptoms

Non-immune-mediated haemolytic anaemia

The inherited causes of haemolysis covered previously are associated to some degree with non-immune mediated haemolysis.

Other causes include infection, trauma e.g. mechanical heart valves, drugs, and thrombotic microangiopathies. Examples are listed in the table below:

Paroxysmal Nocturnal Haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired, haemolytic anaemia, characterised by intravascular haemolysis and is associated with bone marrow hypoplasia. It occurs due to an absence of glycosylphosphatidylinositol (GPI)-anchored proteins on the RBC membrane, which when present, inhibit formation of C3 convertases and prevent terminal polymerisation of the membrane attack complex, thereby protecting the cell from destruction. The absence of these GPI-anchored proteins, CD55 and CD59, leads to complement-mediated destruction of RBCs and WBCs. PNH is a thrombophilic condition and so can be complicated by venous (and less commonly arterial) thrombosis.

Its name comes from the association in some patients with episodic red discolouration of the urine due to the presence of haemoglobin and haemosiderin in the urine from haemolysed red blood cells. The discolouration is more pronounced in the mornings when the urine is more concentrated, hence the name paroxysmal nocturnal haemoglobinuria.

Clinical features – anaemia, fatigue, dyspnoea, haemoglobinuria, thrombosis e.g. symptoms of Budd-Chiari syndrome.

Laboratory bloods – low haemoglobin, may be low WCC or platelets, raised reticulocytes, low haptoglobin, raised lactate dehydrogenase, raised unconjugated bilirubin, consider D-dimer.

Other tests – Flow cytometry for CD55 and CD59 on RBC and WBC has now superseded the historical ‘Ham’s acid haemolysis test’. Fluorescein-labelled proaerolysin (FLAER) is an increasingly used test which is currently the most sensitive and accurate test for diagnosing PNH.

Direct Antiglobulin Test – negative, as the haemolysis is not caused by antibodies.

Management – Allogeneic bone marrow transplant is the only cure but is associated with significant morbidity. Eculizumab is a monoclonal antibody which can be used in certain cases which improves anaemia and quality of life. Other management includes RBC transfusion if required and treatment of thrombosis.

Microangiopathic haemolytic anaemia (MAHA)

Disseminated Intravascular Coagulation (DIC)

DIC is a high mortality, hypercoagulable state due to activation of the coagulation cascade, fibrin and platelet thrombi formation in small vessels, and RBC shearing on fibrin strands. DIC is precipitated by sepsis, trauma, malignancy, and obstetric haemorrhage. It is a consumptive state where bleeding occurs when clotting factors and platelets are used up as part of the hypercoagulable process.

Laboratory findings – thrombocytopenia, prolonged partial prothrombin time and prothrombin time, raised D-dimer, low fibrinogen

Treatment – treat underlying cause e.g. antibiotics in sepsis, heparin for thrombotic predominant patients, replenishment of clotting factors (using fresh frozen plasma) and platelets in patients with a bleeding source or who require interventions with a high bleeding risk

Thrombotic Thrombocytopenic Purpura (TTP)

TTP is another MAHA associated with significant morbidity and mortality. It is due to either an inherited defect or acquired antibody to the ADAMTS13 enzyme which cleaves large von Willebrand Factor (vWF) multimers. Without effective action of the ADAMTS13 enzyme, these large vWF multimers cause platelet aggregation and thrombi formation in small blood vessels. This process results in mechanical RBC fragmentation and thrombocytopenia.

Clinical presentation – Purpura, neurological disturbance (e.g. TIA/stroke, seizures, focal neurology), acute kidney injury (not as severe as HUS), fever

Laboratory bloods – Low platelets, normal coagulation, normal white cell count, raised LDH and bilirubin

Blood film – Schistocytes

Schistocytes on a blood film.

Image Source: Wikimedia Commons (2013). Blood smear with typical schistocytes in TTP marked in blue, Licensed under a creative commons licence. Available for free at: https://commons.wikimedia.org/wiki/File:Blood_smear_with_typical_schistocytes_in_TTP_marked_in_blue_1.tif (Accessed on: 10 January 2023).

Management – Plasma exchange (within 24 hours of presentation), corticosteroids and rituximab

Haemolytic Uremic Syndrome (HUS)

Acute infection by Shiga toxin producing bacteria (namely E. Coli O157:H7) characterised by the triad of thrombocytopenia, acute kidney injury and microangiopathic haemolytic anaemia. HUS usually occurs in children but can present in adults.

Clinical features – nausea and vomiting, bloody diarrhoea, haematuria, weakness, abdominal pain

Laboratory bloods – Anaemia, thrombocytopenia, AKI (raised urea, creatinine, low eGFR), raised reticulocytes, raised LDH, raised bilirubin, low haptoglobin, negative DAT, normal PT and PTT, proteinuria

Bloods film – Schistocytes

Management – Supportive (intravenous fluid administration). Avoid use of antibiotics as it can worsen HUS (unless septic).