Immune-mediated haemolytic anaemia

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Antibody and/or complement-mediated destruction of circulating red blood cells is known as immune-mediated haemolytic anaemia. This chapter covers clinical signs; diagnosis; treatment and prognosis.

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6.1 Mechanisms of RBC destruction in IMHA. In extravascular haemolysis, antibody and complement-coated RBCs are removed from the circulation by macrophages in the spleen and liver. Macrophages carry surface Fc receptors (FcR) for antibody and complement C3b receptors (C3bR) that enhance this interaction. In intravascular haemolysis, the binding of antibody and complement proceeds to formation of the membrane attack complex of the terminal pathway of the complement system. Multiple transmembrane pores form in the RBC membrane; these permit a net influx of ions and water. The swollen RBC ruptures (osmotic lysis), releasing haemoglobin into the circulation. This generally occurs on a massive scale and is associated with the onset of icterus and haemoglobinuria.
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6.2 This dog has severe acute onset immune-mediated haemolytic anaemia (IMHA) with marked tissue jaundice. Sudden intravascular haemolysis has overwhelmed the capacity of the liver to conjugate bilirubin, leading to hyperbilirubinaemia. The dog also probably has haemoglobinuria.
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6.3 The in-saline agglutination test. A drop of EDTA blood from a dog with suspected IMHA is placed on a microscope slide. Small flecks within the sample indicate the presence of probable agglutinates of RBCs. An equal volume of saline is added to the drop of blood. The slide is rocked gently and after a few seconds the aggregated RBCs remain present. This indicates agglutination rather than rouleaux formation and indicates a positive in-saline agglutination test.
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6.4 Formation of spherocytes. An antibody-coated red blood cell (RBC) passes through the spleen and encounters a macrophage. Following interaction with the Fc receptor, the macrophage is unable to engulf (phagocytose) the RBC entirely, but instead removes a portion of the cell membrane. The damaged RBC ‘repairs’ itself by closing over the defect, leading to formation of a smaller cell that lacks the classical zone of central pallor typical of a canine RBC. This cell passes back into the circulation where it appears as a spherocyte.
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6.5 Blood smear from a dog with IMHA. The anisocytosis, polychromasia, spherocytosis (blue arrows) and nucleated erythrocytes (black arrow) are all highly indicative of IMHA.
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6.6 Principle of the Coombs’ test. A suspension of washed antibody- and/or complement-coated red blood cells (RBCs) from the patient is incubated with an antiserum specific for one or more of the immunological molecules that coat the surface of the RBCs (i.e. IgG, IgM or complement C3). In this example, rabbit antiserum specific for canine IgG binds the canine IgG molecules coating the patient RBCs, leading to formation of a lattice-like arrangement that appears grossly as agglutination.
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6.7 The Coombs’ test is performed in a microtitration system. In this plate, four different antisera are titrated across the plate from left to right. Row A contains polyvalent canine Coombs’ reagent, row B contains anti-dog IgG, row C contains anti-dog IgM and row D contains anti-dog complement C3. The antisera are most concentrated on the left side, and with each doubling dilution made towards the right side of the plate they become more dilute. Row E contains saline as a negative control. An equal volume of a suspension of washed patient RBCs is added to each well and the plate is incubated (duplicate plates at both 4 and 37°C). A positive reaction (agglutination) appears as a diffuse mat of RBCs and a negative reaction as a button of RBCs at the base of the well. The titre of the reaction is the inverse of the dilution of antiserum present in the last well of the series that gives a positive result. This dog is Coombs’ positive and the RBCs are coated with IgG (titre 160).

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