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Haemolytic Screen

The Haemolytic Screen

February 17th, 2021


Haemolysis is the destruction of red blood cells prior to the end of their normal 120-day lifespan, and should be considered in anaemic patients with risk factors or no obvious cause of anaemia. Serum markers may be used to diagnose haemolysis and suggest a cause.
Concerns may be raised for the presence of haemolysis in patients with  anaemia, reticulocytosis or hyperbilirubinaemia.
  • The following are considered the basic haemolysis screen:
  • Full blood count - reduced haemoglobin indicates anaemia
  • Blood film - assessing for schistocytes, spherocytes, keratocytes or other poikilocytosis
  • Liver function tests - unconjugated hyperbilirubinaemia
  • Haptoglobin - reduced, particularly in intravascular haemolysis
  • Lactate dehydrogenase (LDH) - elevated in haemolysis
  • Reticulocytes - elevated in haemolysis
  • Direct antiglobulin test (DAT) - positive in immune haemolysis.

Causes of Haemolysis

Haemolysis can be caused by a wide variety of immune and non-immune conditions.
  • Causes of Haemolysis

  • Immune

  • Warm autoimmune haemolytic anaemia - idiopathic, lymphoproliferative disorders, infection, autoimmune disorders, methyldopa, penicillins, cephalosporins, rifampicin, quinine, paracetamol, ibuprofen
  • Cold agglutinin haemolysis - idiopathic, mycoplasma pneumoniae, EBV, lymphoma, paroxysmal cold haemoglobinuria
  • Alloimmune haemolysis - transfusion of mismatched red blood cells, haemolytic disease of the newborn, post stem cell transplant
  • Drug-induced haemolysis
  • Red Cell Fragmentation

  • Microangiopathic haemolytic anaemia - TTP, HUS, DIC, preeclampsia / HELLP, SLE, malignant hypertension, brown snake bite
  • Mechanical haemolysis - valvular leaks, cardiac prostheses, renal replacement, ECMO, march haemoglobinuria
  • Inherited Intrinsic RBC Disorders

  • Enzymopathies - G6PD deficiency, pyruvate kinase deficiency
  • Membranopathies - hereditary spherocytosis
  • Haemoglobinopathies - sickle cell disease, thalassaemias
  • Other

  • Infection - severe sepsis, malaria, babeosis, Clostridium toxin, Meningococcus
  • Hypersplenism
  • Heavy metals - copper (Wilson's disease), lead
  • Paroxysmal nocturnal haemoglobinuria
  • Hypophosphataemia
Thrombotic microangiopathies such as thrombotic thrombocytopaenic purpura (TTP), haemolytic-uraemic syndrome (HUS) and disseminated intravascular coagulation (DIC) are important causes of haemolysis and should not be missed. HELLP syndrome (haemolysis, elevated liver enzymes, low platelets) may occur in pregnant patients. Mechanical haemolysis may be caused by severe valvular leaks, mechanical valves and other cardiac prostheses, by dialysis or ECMO circuits, or by repetitive trauma (e.g. by prolonged marching).
Autoimmune haemolytic anaemias may be either warm or cold. Their causes are wide ranging and include infections, autoimmune disorders, lymphoproliferative disorders and drugs. Alloimmune haemolysis may occur following mismatched blood transfusion, in newborns, or post stem cell transplantation. Certain drugs may also cause immune haemolysis.
Severe infections, malaria, meningococcal and Clostridium infections can cause haemolysis. Certain heavy metals such as copper and lead are also implicated. The syndrome of hypersplenism is a common cause in patients with liver disease, CCF or certain other conditions.
Inherited red blood cell disorders such as hereditary spherocytosis, sickle cell anaemia and thalassaemias are more rare. Glucose-6-phosphate dehydrogenase deficiency may cause oxidative haemolysis and is more common in males of African or Mediterranean descent.

Markers of Haemolysis

  • When ordering a haemolytic screen, start with the following:
  • Full blood count - reduced haemoglobin indicates anaemia
  • Bilirubin (liver function tests) - unconjugated hyperbilirubinaemia
  • Haptoglobin - reduced
  • Lactate dehydrogenase (LDH) - elevated
  • Reticulocytes - elevated
  • Haemoglobin

  • Reduce haemoglobin indicates anaemia, which may be caused by a variety of conditions. Haemolytic anaemia tends to produce a normocytic, normochromic anaemia, and there is often a raised red cell distribution width due to the presence of reticulocytes.
  • Bilirubin

  • Following haemolysis, haemoglobin is metabolised to haem and then bilirubin, which overwhelms the liver's capacity to conjugate it. 
Elevated unconjugated bilirubin is an indicator of haemolysis though can be caused by other disorders including resorption of a large haematoma, certain medications, inherited disorders, acute hepatitis or cirrhosis. As such it is important to also look at the remainder of the liver function panel when interpreting the bilirubin.
  • Haptoglobin

  • Haptoglobin is an alpha-2 glycoprotein secreted mainly by the liver. It binds plasma free haemoglobin following red cell lysis, and as such the haptoglobin level will be reduced in the presence of haemolysis (particularly intravascular haemolysis). 
A reduced haptoglobin may also occur in the context of liver disease due to reduced production. As haptoglobin is an acute phase reactant, it may be increased in infection, inflammation, malignancy or trauma.
  • Lactate Dehydrogenase

  • Lactate dehydrogenase (LDH) is an enzyme present in the cytoplasm of most cells that catalyses the conversion of lactate to pyruvate. 
It is a non-specific marker of cell damage affecting any tissue, and is elevated in haemolysis. Other causes of elevated lactate dehydrogenase include infarction of any kind, malignancy, cirrhosis, trauma, shock and hypoxia.
  • Reticulocytes

  • Elevated reticulocytes are non-nucleated immature red cells. Reticulocytosis (elevated reticulocytes) is indicative of increased erythropoiesis; this may be due to haemolysis, though may also be present following recovery from anaemia or in the context of an increased red blood cell requirement.

Intravascular Haemolysis

  • Haemolysis may occur intravascularly or extravascularly; the latter refers to haemolysis in the spleen or liver. If intravascular haemolysis is suspected, several tests can be performed:
  • Plasma free haemoglobin - elevated
  • Urine haemoglobin - elevated
  • Urine haemosiderin - elevated
  • Plasma & Urine Free Haemoglobin

  • Haemoglobin usually resides within red blood cells, and therefore if the amount of free haemoglobin in plasma is increased then this suggests haemolysis that is occuring intravascularly. 
Similarly, this free haemoglobin will be excreted in the urine; this will result in a dipstick that is positive for blood but with no red blood cells on microscopy.
  • Urine Haemosiderin

  • Urine haemosiderin is the most sensitive test for intravascular haemolysis. The iron that builds up from haemolysis is stored as haemosiderin, which is then excreted - this results in brownish urine with an excess of iron on iron staining.

Immune vs Non-Immune Haemolysis

The direct antiglobulin test (DAT), also known as the direct Coomb's, tests for IgG and complement 3 (C3) on the surface of red blood cells. The test is performed by adding antibodies against human IgG or C3 and monitoring for agglutination.
A positive direct antiglobulin test indicates an immune (autoimmune, alloimmune or drug-induced) cause of haemolysis, and may be positive before haemolysis occurs.
For a list of immune and non-immune causes of haemolysis, see the table at the top of the page.

The Blood Film in Haemolysis

Abnormal red blood cell morphology may provide clues regarding the presence or cause of haemolysis.
  • The Blood Film in Haemolysis
  • Schistocytes

  • Schistocytes are small, irregular fragments of red blood cells of varying shapes. These are a sign of microangiopathic haemolytic anaemias such as TTP, HUS, aHUS, DIC or HELLP syndrome; they may also occur with mechanical haemolysis such as due to cardiac prostheses, dialysis or extracorporeal membrane oxygenation (ECMO).
Microspherocytes are counted as schistocytes - they are small round cells with loss of central pallor.
  • Spherocytes

  • Spherocytes are round cells with loss of central pallor, and occur due to removal of a portion of the cell membrane by phagocytes. Spherocytosis may be hereditary, though are also a sign of haemolytic anaemia.
  • Prekeratocytes & Keratocytes

  • Keratocytes, or bite cells, are cells with bite-like defects in their membranes, due to phagocytosis of a Heinz body. Keratocytosis may occur in the context of oxidative haemolysis, in the context of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency with a trigger (particularly medications) or in patients with unstable haemoglobins.
Prekeratocytes, also known as blister cells, are similar to bite cells with a small sliver of membrane remaining over the bite.
  • Sickle Cell

  • Sickle cells, also known as drepanocytes, are crescent-shaped cells caused by aggregation of haemoglobin S. These are pathognomonic of sickle cell disease.
  • Polychromasia

  • Polychromasia refers to macrocytic erythrocytes with a bluish tinge - these cells are reticulocytes, which are immature red cells. This finding is an indicator of haemolytic anaemia, though polychromasia may also occur following haemorrhage or during recovery following bone marrow suppression.
  • Red Cell Inclusions

  • The Blood Film in Haemolysis
Nucleated red blood cells are immature red blood cells that contain nuclei. They may present in the context of severe anaemia, severe sepsis, hypoxia or bone marrow disease.
Heinz bodies are basophilic denatured haemoglobin molecules that are present in the context of oxidative haemolysis. These may only be seen on supravital staining.
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