Erythroid system

• What are reticulocytes?

  Young (immature/non-nucleated), erythrocytes prematurely released to blood from the bone marrow in regenerative anaemias.

  To visualise:

  • New methylene blue precipitation demonstrates RNA-protein Complexes.
  • Young red cells including reticulocytes have ‘polychromatophil” appearance on Romanowsky stain.

  Clinical applications:

  • evaluation of erythropoiesis in bone marrow.
  • Differentiation of regenerative and non-regenerative anemia.

  
• Evaluating reticulocytes

  Reticulocyte counts:

  • Manual
  • Automated (some haematological analysers)

  Absolute reticulocytes count (ARC)

  = observed % reticulocytes x RBC (x10^12/l) x 10

  • Independent of variation of RBC numbers.

  With a more severe anaemia you expect to see a larger number of reticulocytes than with a mild anaemia.
• Species variation in Reticulocyte response

  Dogs:

  • Low number of reticulocytes (<1%).
  • Expect at least (>60x10^9/L) in regenerative anaemia.

  Cats:

  • Low number or Reticulocyte (0.2-1.6%)
  • Cats have two morphological types of reticulocytes:
  • Aggregate blue stained coarse clumping (0.5% of erythrocytes)
  • Punctuate small, blue stained dots (1-10%)

  Kinetics of Aggregate vs Punctuate means we only consider aggregate in assessment of regeneration.

  Ruminants and horses:

  • Virtually no reticulocytes in normal blood;
  • Reticulocytes may not appear even in severe anaemia in horses
• Blood morphology in dogs

  • Larger erythrocytes
  • Uniform size
  • Central pallor

  
• Blood morphology in cats

  • Smaller erythrocytes.
  • Anisocytosis (variation in size)
  • Scarce central pallor (less concave)

  
• Blood morphology variations within dog breeds

  Macrocytosis in some poodles.

  Akitas have unusually small erythrocytes & particularly high potassium content.

  Greyhounds have high PCVs (0.55-0.6L/L)
• Size of red blood cells (RBCs)

  Measure of RBC size:

  • MCV - mean corpuscular volume
  • PCV (L/L) x1000/ RBC count

  Cell volumes measured directly and averaged or by calculation depending on analyser.

  Normocytosis - normal range of RBC size

  Macrocytosis - regenerative anaemia. FeLV infection, myeloproliferative disease.

  Microcytosis - Iron deficiency
• Red cell distribution (RDW)

  A numeric representation of the variability in RBC size. More sensitive than MCV.

  Normal: similar RBC sizes.

  Anisocytosis: unusual large variation in RBC size, e.g. if large numovers of Microcytes or macrocytes (increased RDW).
• Macrocytosis

  Normal breed-related finding in poodles.

  Commonly seen in damaged or aged samples - artefact or erythrocyte swelling.

  Hypochromic macrocytosis - regenerative anaemia (haemorrhage, haemolysis).

  Normochromic macrocytosis (non-regenerative) - associated with feline leukaemia virus infections in kittens and myeloproliferative disorders in dogs and cats.
• Microcytosis

  Hypochromic Microcytosis - marker of altered iron metabolism.

  • Iron deficiency - most common cause in dogs: occult blood loss from GI tract; neonatal piglets; blood loss through internal or external parasites.
  • Dogs/cats with Porto systemic shunts (liver defect) - effect on iron metabolism currently unknown

  Akitas have unusually small erythrocytes.
• Colour of RBC

  MCH and MCHC:

  • Mean cell haemoglobin and Mean cell haemoglobin concentration.
  • MCHC (g/L) = Hb (g/L)/PCV(L/L) - more useful indicator than MCH since cell size is taken into account.

  Indicated the average concentration of Hb in RBCs:

  • Normochromic - normal MCHC = normal hg content
  • Hypochromic - Low MCHC = low hg content.

  Rasied MCHC is almost always die to haemolysis (intravascular or sample handling) or lipaemia.
• Polychromasia

  Blue-ish/grey colouration of large RBCs on a Giemsa stained blood smear.

  In dogs, an average of >10 polychromatic red cells per oil immersion field suggests a marked regenerative response.

  Some analysers can give a HDW (haemoglobin distribution width - the Hgb equivalent of RDW).
• MCV, MCH and MCHC

  MCV: mean cell volume;

  • Average red cell size.

  RDW: red cell distribution width

  • Degree of variation on red cell size
  • Could be more small cells or more large cells or both.

  MCHC: mean cell haemoglobin concentration

  • Average haemoglobin concentration in cells
• Shape of RBC

  Poikilocytosis:

  • Alteration in cell shape.
  • Abnormal erythropoeisis
  • Specific organ dysfunction
  • Codocytes (target cells)
  • Acanthocytes
  • Spherocytes
  • Schistocytes

  Artefacts: cremation - burr cells
• Codocytes or ‘target cells’

  Appearance of a target with a bullseye;

  Central, haemoglobinised area surrounded by an area of pallor; periphery of the cell contains a band of haemoglobin.

  Lacking normal biconcave cross section due to folding of cell membrane.

  Seen in iron deficiency anaemia, liver disease with cholestasis and after splenectomy of dogs. Often of little significance.

  
• Acanthocytes (‘spur cells’)

  Rounded projections of variable diameter and length, unevenly distributed.

  Due to increase in membrane cholesterol or in association with RBC fragmentation.

  Seen in diffuse liver disease, splenic haemangiosarcoma, haemangioma or Porto-systemic shunts.
• Spherocytes

  Small, densely staining spherical RBCs, lack central pallor.

  Small portion of the cell membrane may have been phagocytosed by mononuclear phagocytes; residual cellular tissue resumes the smallest shape possible - a sphere.

  Rarely recognised in cats since normal RBCs have less of central pallor than in dogs.

  Presence implies that erythrocytes have surface bound antibodies or complement.

  Indicator of immune-mediated haemolytic anaemia in dogs.

  
• Schistocytes

  Irregular, fragmented erythrocytes.

  Through mechanical trauma to circulating erythrocytes.

  The erythrocytes flowing in the blood split in two.

  Markers of disseminating intravascular coagulation (DIC) and other angiopathies.

  
• Crenation ‘Burr cells’

  RBCs with spiked projections of more uniform length.

  Echinocytes

  Some normal in ruminants

  Rarely snake envenomation

  Occasionally in dehydration

  
• Rouleaux formation

  Clustering, sticking, piling of RBCs.

  Normal finding in horses.

  Indicated inflammation in small animals

  Relates to increased ‘stickiness’ of plasma with increased globulin content.

  
• Agglutination

  Can be seen microscopically and macroscopically.

  Seen in immune mediated haemolytic anaemia and mismatched blood transfusions.

  
• Heinz bodies

  Irregular shaped, refractile inclusions.

  Consisting of oxidative denatured haemoglobin.

  Increased numbers in paracetamol and onion toxicity and other oxidative compounds; more common in cats than in dogs.

  Up to 10% of RBCs in normal cats - can be caused by stress.

  In cats also commonly associated with diabetes mellitus, lymphoma and hyperthyroidism.
• Basophillic stippling

  Multiple, small, dark blue, punctuate aggregates in RBC.

  In Vivo aggregation of ribosomes into small basophillic granules.

  Caused by low levels of pyramidine 5’-nucleotidease enzyme that catabolises ribosomes.

  In cats (more common) and dogs associated with intensely regenerative anaemia.

  Associated with lead poisoning

  Normal in immature erythrocytes in ruminants.

  
• Nucleated erythrocytes

  Erythrocytes with remains of a nucleus.

  Regenerative anaemia:

  • Early release of RBCs from bone marrow and extra-medullary haematopoiesis sites in response to hypoxia.

  In absence of anaemia:

  • Non-functioning spleen.
  • Marrow damage
  • In cats, in absence of polychromasia, indication of myelodysplasia or myeloproliferative disease.

  
• Howell-Jolly bodies

  Refractile, single dark blueish bodies in RBCs of variable size.

  Repressing nuclear remnants.

  Regenerative anaemia:

  • Splenectomy
  • Suppressed splenic function
  • Higher percentage seen in normal cats.

  
• Babesiosis

  Is a Protozoa.

  Tick born disease.

  Intracellular.

  Endemic in cattle.

  Dogs: Babesia canis or B. gibsoni, rare in UK, imported.

  Uncomplicated or complicated forms:

  • Haemolytic anaemia.
  • Systemic inflammatory response syndrome (SIRS), multiple organ disfunction syndrome (MODS).
• Mycoplasma Haemofelis

  Bacterial infectious agent.

  • Highly pelomorphic, appearing as chains, discs or rods.\On surface or embedded into RBC membranes.
  • Worldide distribution.
  • Diagnosis confirmed by PCR.

  Haemobartonellosis or feline infectious anaemia.

  Variable clinical signs including regenerative anaemia, pyrexia and malaise.
• Misleading results from an analyser

  High MCHC:

  • Not physiological to cram more Hgb into red cells than they will take.
  • Haemolysis (sample handling or intravascular).
  • Lipaemia

  MCV:

  • Swelling of transport.
  • Mis-identification - pairs and triplets, cross over with large platelets.
  • Cell shrinkage or expansion in sample e.g. hyperosmolar.
  • Will impact on calculated PCV/HCT.
• Erythron

  When evaluating the erythron:

  • Is there inadequate, adequate or excessive red cell mass to deliver oxygen to tissues?
  • Is there evidence of anaemia?
  • Is there evidence of regeneration?
  • What is the cellular character of the anaemia?
  • Normocytic, normochromic, hyperchromic, Macrocytic.
  • Is there evidence of polycythaemia?
  • Relative or absolute.
• Packed cell volume, red cell count, Hgb

  PCV (HCT), RBCC and Hgb.

  All three are measures of red cell mass anf oxygen carrying capacity.

  Usually interpret them as a block.

  All equally affected by haemoconcentration.

  Will usually increase and decrease in line with one another.

  When they are discordant find out why
• Classification of anaemia

  Based on MCV and MCHC:

  • Blunt measure
  • Microscope visible findings may not be sufficient to push parameter out of reference range.
  • Machine dot-plots and histograms more sensitive.

  Normocytic normochromic:

  • Often aneamia of illness or pre-regenerative or occasionally non-regenerative.

  Macrocytic hypochromic:

  • Classic highly regenerative.
  • SometInes could just be cell swelling of transport.

  Microcytic hypochromic:

  • Classic iron deficiency - chromic external blood loss.
  • Without anaemia - portosystemic shunts
• Polycythaemia

  Increase in PCV, Hgb concentration and RBC count.

  Term polycythaemia implies increased number of severa haemopoetic cell lines (human), however dogs and cats with polycythaemia Vera usually have normal neutrophil and platelet counts.
• What is Relative polycythaemia?

  Apparent increase in RBC due to a decrease in fluid in circulation (often increased total protein and albumin).

  PCV is increased but no increase in RBC production.

  • Dehydration (water or acellular fluid loss):
  • E.g. vomiting, diarrhoea, polyuria, extensive burns, adipose a, water deprivation.
  • Exercise, fear, excitement, severe pain - stress:
  • Adrenaline secretion, splenic contraction and transient redistribution or RBC from the spleen to the circulation.

  Resolves after rehydration or removal of cause of splenic contraction.
• What is absolute polycythaemia?

  True increase in RBC mass die to increased RBC production/ release (usu polychromasia, anisocytosis and reticulocytes).

  Primary polycythaemia:

  • Rare myeloproliferative disorder.
  • Abnormal response of RBC precursors.
  • Normal EPO levels.

  Secondary polycythaemia

  • Chronic tissue hypoxia of renal tissues (low arterial pO2) due too:
  • Heart/lung diseases, high altitude, thrombosis, constriction of renal vessels.
  • Renal tumour or cysts