Non-Immune Hemolytic Disorders

Non-Immune Hemolytic Disorders (NIHDs)

• NIHDs are characterized by positive routine hemolytic tests, but a negative anti-human immunoglobulin (Coombs) test.
• Direct and Indirect Antiglobulin Tests are negative
• NIHDs are broadly classified into hereditary and acquired forms.

Hereditary Non-Immune Hemolytic Disorders

• These are mainly intra-corpuscular
• Includes:
  • Membranopathies
  • Haemoglobinopathies
  • Enzymopathies
  • Rare hereditary Thrombotic Thrombocytopenic Purpura (TTP)
• Presents with intravascular or extravascular haemolysis.

Acquired Non-Immune Hemolytic Disorders

• These are mainly extra-corpuscular
• Includes:
  • Red cell fragmentation
  • March haemoglobinuria
  • Infections
  • Chemical and physical agents
  • Paroxysmal nocturnal harmoglobinuria (PNH)
  • Acquired disorders of red cell membrane
• Presents with intravascular haemolysis.

Acquired Non-Immune Hemolytic Disorders Classification

• Red cell fragmentation syndrome/Mechanical Haemolytic Anaemias
• March haemoglobinuria
• Infections
• Chemical and physical agents
• Paroxysmal nocturnal haemoglobinuria
• Acquired disorders of red cell membrane

Red Cell Fragmentation Syndrome/Mechanical Hemolytic Anemias

• The syndrome is due to physical damage to red cells in circulation.
• Characterized by the presence of Schistocytes
• Schistocytes or Schizocytes are fragmented red cells that can take on different shapes, like triangular, helmet, or comma-shaped with pointed edges, usually more than 2.
• They are usually microcytic without a central pallor area
• They share the deformability of normal red cells, but have a reduced lifespan.
• Schistocytes exist in every person with a typical count of 0.2-0.5% as measured by a peripheral blood film.
• Higher counts are found in Red Cell Fragmentation Syndrome.
• Red Cell Fragmentation syndrome is broadly divided into Macro Angiopathic and Micro Angiopathic Haemolytic Anaemias.

Macro Angiopathic Haemolytic Anaemias/Cardiac Haemolytic Anaemias

• Cardiac Haemolysis typically follows cardiac surgery involving prosthetic valves, patches, or vessels.
• The use of newer materials like homografts/xenografts has made it less prevalent.
• It is rare, occurring in about 3% of patients with prosthetic heart valves.
• Haemolysis is caused by artificial materials and turbulent blood flow, resulting in red cells’ contact and collusion with the material.
• Damage to red cells could happen because of abnormal surfaces:
  • Calcified or stenotic heart valves
  • Artificial heart valves
  • Arterial grafts
• It could also be down Arteriovenous malformations such as in Cavernous haemangioma (Kasabach-Merit Syndrome).

Arteriovenous Malformations (AVMs)

• AVMs happen when blood vessels develop incorrectly
• Arteries and veins become tangled, creating direct connections that bypass normal tissues.
• Typically occurs during development before birth or shortly after.
• Usually, no capillaries are present so Blood flows directly between arteries and veins, usually at high velocity.
• It can occur anywhere in the body

Micro Angiopathic Haemolytic Anaemias (MAHA)

• MAHA is a clinical syndrome defined as intravascular haemolysis with red cell fragmentation which is caused by the destruction of red cells in abnormal micro-circulation.
• Three lesions can give rise to MAHA:
  • Presence of fibrin strands in circulation
  • Platelet aggregation
  • Vasculitis

Fibrin Strands Presence in Circulation

• Red cells fragment as they pass through fibrin strands
• It is common in:
  • Disseminated Intravascular Coagulopathy (DIC)
  • Meningococcal sepsis
  • Carcinomatosis

Platelet Aggregation

• Platelets adhere to small vessels, forming localized or generalized aggregates
• Commonly seen in Thrombotic Thrombocytopenic Purpura (TTP).
• Haemolytic Uraemic Syndrome.
• Homograft rejection (micothrombi in transplanted organ)

Vasculitis

• Endothelial surfaces become roughened from inflammatory reactions so red cells fragment when they collide with the roughened endothelial cells
• Seen in:
  • Polyarteritis nodosa
  • Systemic Lupus
  • Wegener’s granulomatosis
  • Renal cortical necrosis
  • Acute glomerulonephritis

Haemolytic Uraemic Syndrome

• HUS is an acquired disorder mainly affecting infants and children under 4 years old.
• A syndrome is rare after puberty and cases can be sporadic or epidemic
• Classically presents with a triad of intravascular haemolysis, renal failure, and thrombocytopenia.
• First described by Gasser in Europe, Brain et al. highlighted red cell fragments, diffuse intravascular coagulation, and microangiopathic haemolytic anaemia.
• The cause is unknown or not fully understood, but host and environmental factors are implicated.
• Host Factors:
  • Abnormal reduced creation of prostacyclin; Prostacyclin is normally produced by muscles and endothelium as a vasodilator and anti-platelet.
  • Only after platelets have adhered to the endothelium is Thromboxane A2, another prostaglandin, created.
  • Thromboxane A2’s physiological properties are opposite to prostacyclin.
  • Occurrence in different family members suggests constitutional and genetic factors.
• Environmental Factors:
  • Can be associated with infective agents like viruses, rickettsia, and bacteria.
  • Epidemic forms commonly involve infection with Eschericia coli O157 (verotoxin producing).

HUS Clinical Features

• Diarrhoea is the most common symptom, often bloody, and is associated with vomiting
• Symptoms usually last for a day or 2 weeks before being followed by:
  • Haemoglobinuria
  • Proteinuria from acute renal failure
  • Uraemia
  • Anaemia
  • Thrombocytopenia
  • Jaundice
  • Possible CNS signs, such as convulsion or coma

HUS - Laboratory features

• Lots of red cell fragments on peripheral blood
• Anisocytosis, Poikilocytosis
• Polychromatic cells, Nucleated red cells
• Severe thrombocytopenia
• PT, PTTK, and TT are usually normal

HUS - Management

• Management is mainly supportive pending renal failure
• Red cell transfusion
• Platelet transfusion rarely needed except there is bleeding
• Coagulation factors replacement with Fresh Frozen Plasma (FFP) if there is evidence of DIC.
• Corticosteroids and platelet aggregation inhibitors

Thrombotic Thrombocytopenic Purpura (TTP)

• A clinical syndrome is characterized by a pentad:
  • Fever
  • Anaemia
  • Thrombocytopenia
  • Neurological abnormalities
  • Renal Failure
• As with HUS, there is platelet aggregation in small blood vessels
• Aetiology is unknown or not fully understood

TTP - Possible Aetiology

• The disorder can be congenital or acquired.
• There is always abnormal platelet aggregation to small vessels due to a persistent presence of unusually large von-Willibrand Factor (vWF)
• The vWF is normally degraded by a serum metaloprotease called ADAMTS13 or vWF cleaving protein (vWF-cp). Note the ADAMTS13 gene is located on chromosome 9q34
• ADAMTS13 is predominantly produced by hepatocytes and also by vascular endothelial cells, although in limited quantities
• ADAMTS 13 level is significantly reduced or completely absent in TTP

TTP - Aetiology: Congenital vs Acquired forms

• Mutations at the ADAMTS13 gene in the congenital form
• Patients with the acquired form develop autoantibodies against ADAMTS13.
• Aetiology can be serum factors deficiency that promote prostacyclin production by endothelial cells.
• Prostacyclin inhibits both platelet adhesion and aggregation to the endothelial cell

TTP - Clinical Features

• More common amongst teenagers and young adults
• Rare but a devastating disease with a a mortality rate of up to 50% in conservatively managed cases.
• Affects both sexes equally
• Onest of fever is sudden
• Fever and neurological abnormalities commonly predominate, convulsion, coma, paralysis, psychiatric manifestations like hallucination.
• Purpura may or may not be early.

TTP - Clinical and Laboratory Features

• Anaemia
• Marked haemoglobinuria
• Jaundice
• Renal failure is not generally seen
• Fragmented and contracted red cells are seen
• Polychromasia
• Thrombocytopenia
• Proteinuria is seen in all cases, although it may be intermittent.

TTP - Management

• Plasmapharesis: Plasma exchange via plasmapharesis, replacing Fresh Frozen Plasma + Plasma Proteins + Platelets
• Prostacyclin analogues: Infusion may be benefit especially if there is response to FFP
• Splenectomy
• Removal of stimuli with broad-spectrum antibiotics or termination of pregnancy

March Haemoglobinuria

• Haemoglobinuria following running has been known for over 100 years
• It is caused by damage to red cells between the small bones of the feet during prolonged marching or running
• Seen in joggers and is benign, with no associated red cell fragments on blood film.

Infections Causing Haemolytic Anaemia

• Acute haemolytic anaemia of G6PD deficiency
• Micro angiopathic haemolytic anaemia, e.g pneumococcal and meningococcal septicaemia.
• Malaria causes haemolysis by extra vascular destruction of parasitised red cells or direct intravascular destruction of red cells when the sporozoites break out of red cells.
• Black water fever is an acute intravascular haemolysis associated with acute renal failure during Falciparum malariae infestation.
  • Uncommon but devastating complication
  • The onset is acute with marked haemoglobinaemia, haemoglobinuria and rapid progression to oliguric renal failure
  • Initially described among whites treated with quinine, but reported among blacks in malaria-free areas
  • Linked high parasitaemia and impaired immunity
• Babesiosis: a rare disorder that is caused by the parasitic protozoan, Babesia. - Transmitted by Ixodes ticks - Ixodes vicinus (Europe associated with cattle) - Ixodes dammini (America assocaited with rodents and deer) - Fatal with acute onset in splenectomized patients
• Other infective agents:
  • Toxoplasmosis (Toxoplasma gondii, severe immunosuppression)
  • Oroya fever (Bartonella bacilliformis)
  • Clostridium perfringens
  • Psittacosis and Yersinia enterocolitica
    • Viruses like Dengue, Yellow fever and West African haemorrhagic fever

Chemical and Physical Agents Causing Haemolytic Anaemia

• Dapsone and Salazopyrin cause oxidative damage to red cell membrane, resulting in intravascualar haemolysis
  • Heinz bodies are prominent in red cells
• High levels of cupper in the blood can cause intravascular haemolysis, such as in Wilson’s disease
• Chemical poisoning from lead, chlorate, or arsine can cause haemolysis
• Nitrites (well water and vegetable juices) and Nitrates may cause methaemoglobinaemia in infants

Paroxysmal Nocturnal Haemoglobinuria (PNH)

• Also called Machiafava-Michaelis syndrome
• Rare, acquired, somatic mutation and clonal intracorpuscular disorder of bone marrow stem cells
• There is deficient synthesis of glycosylphosphatidylinositol (GPI) anchor protein on red cell membrane
• GPI attaches surface proteins to red cell membrane
• Generated by mutation in the X chromosome gene that encodes phosphatidylinositol glycan protein A (PIG-A).
• Without GPI as an anchor protein, no GPI-linked proteins can exist, such as CD55 and CD59
• Without CD55 and CD59, there is a lack of Decay Accelerating Factors (DAF) or Membrane inhibitor of reactive lysis (MIRL)
• DAF recognizes C3b and C4b fragments generated when C3 and C4 are activated, accelerating decay of C3 and C5.
• MIRL is a potent inhibitor of MAC
• DAF/MIRL deficiency makes red cells sensitive to lysis by complement, causing intravascular hemolysis

PNH - Clinical Features

• Males and females are equally affected
• Two types exist
  • Classical haemolytic PNH, which is commonly seen
  • Laboratory PNH, detected only by laboratory tests
• Haemolytic PNH is very rare in infants and young children, but is common in adolescent and young adults up to 40 years
• Haemoglobinuria is usually noticed in the first urine after sleep, clearing throughout the day
• Abdominal pain, often severe and intermittent, is unrelated to meals
• Thrombotic complications, particularly in large veins, are common

PNH - Laboratory Features

• Haemosiderinuria occurs constantly
• Iron deficiency may develop
• Splenomegally
• Peripheral blood film exhibits:
  • Anaemia
  • Polychromasia
  • Neutropenia
  • Marked thrombocytopenia
  • Tear drop poikilocytes
  • Low Neutrophil Alkaline Phosphatase (NAP) score.
• NAP score measures the proportion of WBCs containing alkaline phosphatase; normal levels are between 20 and 150 Because WBCs are derived from the clone, they are also deficient in GPI.
• As an Alkaline phosphatase is also an anchor protein, it is measured in the NAP score. MIRL's lack makes the NAP score is low

PNH - Diagnosis

• Confirmed from the following tests:
  • Ham-Dacie Test or Acidified Serum Lysis Test: activates alternate complement pathway, lysing PNH cells via high complement sensitivity
  • Sucrose Lysis Test
  • Thrombin Lysis Test: 2 and 3 activate the classical complement pathway
• Diagnosis is confirmed by Flow Cytometry

PNH - Treatment

• Iron therapy
• Anticoagulation with warfarin
• Immunosuppression
• Bone marrow transplantation
• It typically remits with a median survival of 10 years.

Acquired Disorders of Red Cell Membrane

• The lipids on the red cell membrane are in equilibrium with the plasma lipids
• Changes in the ratio of free cholesterol to phospholipids in plasma can alter cell shape, resulting in haemolysis
• Primarily pathologies in the liver, such as acute or chronic hepatitis and/or liver cirrhosis
• Gilbert’s disease (inherited nonhaemolytic unconjugated hyperbilirubinaemia)
• Ziever’s syndrome presents as intravascular haemolysis and acute abdominal pain; primarily seen in alcoholics
• Wilson’s disease is due to high level of copper ion in the blood and may result in acute intravascular haemolysis with liver and neurological features
• Vit E deficiency can lead to haemolysis, cell contraction, and thrombocytosis and is seen in infants maintained on diet rich in polyunsaturated fatty acids