RBC

What type of tissue is blood? What is it made of?

Blood = Connective tissue with fluid (plasma) and cellular part

Plasma (46 - 63%):

1. Water (92%)

2. Plasma proteins (7%)

3. Regulatory proteins (eg. hormones and enzymes)

4. Other solutes (eg. nutrients, gases and wastes)

Cells (37 - 53%):

1. Leukocytes

   1. Granulocytes (neutrophils, eosinophils, basophils)

   2. Agranulocytes (lymphocytes, monocytes)

2. Erythrocytes

3. Platelets

Describe normal body fluid compartments

• % of TBWT that blood makes up (cat vs. dog)

• % TBWT made of water (+ compartments)

• Movement of fluid between compartments

Blood: 5 - 10% BWT

• Cat = 55mL blood/kg BWT

• Dog = 90mL blood/kg BWT

Water: 60 - 70% BWT is water

• 67% intracellular fluid (ICF)

• 33% extracellular fluid (ECF)

  • Plasma in blood (IVF)

  • Interstitial fluid (ISF)

Movement: Membrane are permeable to water → free exchange until equilibrium reached

Extramedullary haematopoiesis (EMH)

Liver and spleen produce blood cells when the bone marrow does not work

Describe formation of blood cells and what is required

Formation: Derived from primitive (pluripotential) stem cells in the bone marrow

• ALL blood cells have a limited lifespan and are incapable of self-renewal

• Bone marrow produces steady supply of replacements to replace old cells

Requirements: Cytokines stimulate differentiation of the stem cells into committed (unipotential) cell lines forming each blood cell type eg.

1. Erythropoietin (EPO) → RBC production

2. Thrombopoietin

3. Granulocyte and macrophage colony stimulating factors (CSF)

Describe EIGHT organs involved in haematopoiesis (+ functions of each organ)

1. Bone marrow

   1. Main source of blood cells (RBC, WBC and PLT)

   2. Stores iron for heme synthesis

   3. Source of stem cells for T and B lymphocytes to be produced elsewhere

2. Liver = Major haematopoietic organ during 1st ½ of embryonic life (bone marrow takes over BUT retained EMH potential as adult)

3. Spleen

   1. Produces T/B lymphocytes and plasma cells from bone marrow stem cells

   2. Stores RBCs and iron

   3. Removes old RBCs from circulation

   4. Retained haematopoietic potential (EMH)

4. Lymph nodes = Produce T/B lymphocytes and plasma cells from bone marrow stem cells to participate in Ab production and CMI

5. Thymus = Controls differentiation of bone marrow stem cells into T lymphocytes

6. Macrophages of monocyte phagocytic system (MPS) = Macrophages lining the sinusoids of the spleen, liver, lymph nodes and bone marrow complete intracellular haemolysis

7. Kidney = Hypoxia stimulates renal tissue to produce EPO for erythropoiesis

8. Stomach and intestinal mucosa

Describe the differentiation of multipotential haematopoietic stem cell (haemocytoblast)

Erythropoiesis

• Location

• Duration

• Process

• 3 Pools of RBCs in the body

Location: BONE MARROW (also liver and spleen EMH)

Duration: ~7 days from initial stimulation of stem cells by EPO to reticulocyte release

Process:

1. Pluripotential stem cells → Erythroid stem cell

2. Erythroid stem cells → Rubriblasts in response to EPO which begins to produce Hb and other proteins

3. Rubricytes produced from each rubriblast

4. Rubricyte → Metarubricyte (final nucleated stage)

5. Metarubricyte → Reticulocyte with no nucleus (triggered when RBCs reach critical Hb content)

6. Reticulocytes remain in bone marrow for 2 - 3 days

7. Reticulocytes released into circulation for final maturation as a erythrocyte

Pools of RBCs:

1. Erythrocyte precursors in bone marrow

2. Mature erythrocytes in blood

3. Mature erythrocytes in spleen

Regulation of Erythropoiesis

• Stimulus

• 4 Functions of EPO

  • Species-specific responses

• 4 Other requirements

Stimulus: Accelerated RBC production if demand for O2 is great (eg. anaemia) OR hypoxia detected by renal tissue → Stimulates production of EPO in the kidney

Functions of EPO:

1. Stimulates stem cells to differentiate into rubriblasts (instead of other cell lines)

2. Promotes Hb synthesis

3. Reduces maturation time of RBC precursors

4. Stimulate early release of immature reticulocytes

Species Variation: Dogs > Cats > Cows response to EPO

• Horses do NOT release reticulocytes (do not respond to EPO) → Requires bone marrow aspirate to determine regenerative response

Other Factors: Deficiency → Anaemia

1. Essential vitamins (B12, B6, folic acid)

2. Essential minerals (iron, copper, cobalt)

3. Hormones (GH, ILGF, testosterone, thyroid)

4. IL and colony-stimulating factors

Name the following species each RBC belong to (+ description)

DOG Biconcave with obvious central pallor	CAT Smaller than dogs Anisocytosis Less obvious central pallor	GOAT Spherical and irregular shapes No central pallor
DEER Sickle-shaped	CAMELID Elliptical	BIRD, REPTILE, FISH Nucleated and elliptical

Describe the structure of erythrocytes

• Hb content%

• Hb structure

• Heme structure

• Organelles and energy source

• Shape and 2 functions

Content: RBCs = 95% Hb

Structure:

• Hb = 4 globin subunits which are linked and folded about a central heme molecule

• Heme = Protoporphyrin ring with an atom of iron for O2

Energy: No mitochondria or ribosomes → Energy source from glucose in plasma ONLY

Shape: Biconcave →

1. Increase SA to allow more Hb to attach to RBC membrane for gas exchange

2. Assists RBC deformation through small capillaries

Lifespan of 3 different blood cells

1. Erythrocytes (months)

   • Larger the animal → Longer the lifespan

   • Cat = 70d, dog = 120d, horse = 145d, cow = 160d

2. Platelets (days)

3. WBCs (hours and replaced 2x daily)

Extravascular Haemolysis

• Normal function

• Normal MoA of extravascular haemolysis

• Outcomes of the components of RBC breakdown

Function: Old/damaged RBCs that have reached the end of their lifespan are removed from circulation for membrane degradation

• Allows their components to further broken down and recycled (spleen #1)

• aka. intracellular haemolysis (within macrophages of the spleen, liver and bone marrow OUTSIDE blood vessels)

• Physiological process which becomes pathological if occurring at a rapid rate

MoA:

1. Blood flows through the spleen

2. Old/worn out cells have different structure and contain proteins that the splenic macrophage recognises

3. RBCs enter the macrophage's cytoplasm to undergo haemolysis by digestive enzymes

4. Haemoglobin (Hb) is released from lysed RBCs and broken down into heme, globin and iron

Components of RBC Breakdown:

1. Heme = Further broken down into bilirubin and carried by albumin to liver for processing and elimination as bile in faeces

   • Bilirubin = Yellow/orange

2. Globin = Broken down into AA for recycling

3. Iron = Incorporated into haemosiderin (or ferritin) for storage in macrophages

   • Haemosiderin = Yellow/brown

Extravascular vs. Intravascular Haemolysis

• Definition

• Severity

• Signs

• 5 Mechanisms (+ DDx)

EXTRAVASCULAR HAEMOLYSIS (90%)

Definition: Haemolysis that occurs by splenic macrophages NOT within the blood vessels

• Physiological OR pathological

Severity: More chronic than intravascular haemolysis (NO haemoglobinaemia or haemoglobinuria → Hb recycled and NOT lost in urine)

Signs:

1. No haemoglobulinaemia or haemoglobulinuria

2. Hyperbilirubinaemia (overwhelming of liver)

3. Abnormal RBC morphology (eg. erythrocytic parasites, spherocytes, schistocytes)

4. Positive Coomb’s test

5. Splenomegaly

DDx:

1. Reduced RBC deformability due to membrane alteration (eg. blood parasites, immune-mediated anaemias, microangiopathies)

2. Impaired glycolysis or low ATP content in RBCs (eg. pyruvate kinase deficiency)

3. Immunoglobulins/C3 opsonisation of RBC membranes (eg. IMHA)

INTRAVASCULAR HAEMOLYSIS (10%)

Definition: Lysis of RBCs within the lumen of the blood vessels (fragile) resulting in Hb released directly into the plasma

• Usually pathological (small amounts occur normally but bound to haptoglobin)

Severity: More severe than extravascular haemolysis due to urine loss of Hb

Signs:

1. Haemoglobinaemia and haemoglobinuria

2. Decreased PCV, decreased RBC and [Hb]

3. Increased artifactual MCHC (agglutination)

4. Hyperbilirubinaemia (jaundice/icterus) with exceeded liver capacity to remove and conjugate bilirubin + bilirubinuria

5. Altered RBC morphology (eg. Heinz bodies, schistocytes and parasites)

6. Positive Coomb’s test

7. Decreased serum haptoglobin concentration (protein binding free Hb and complex removed by kidney)

DDx:

1. Complement-mediated lysis (eg. immune-mediated anaemia)

2. Oxidative injury → Denaturation of Hb to Heinz bodies and lysis of RBCs (eg. chemical and plant toxins)

3. Osmotic lysis due to RBC membrane alterations, infusion of hypotonic fluids or drinking lots of water

4. Erythroparasites

5. Leptospirosis and clostridium

Pathology of haemoglobinuria due to intravascular haemolysis

• What occurs normally to free Hb?

• What occurs when in excess?

1. Some free Hb dissociates into globin dimers which binds haptoglobin in plasma to form the haptoglobin-haemoglobin dimer complex

2. Hpt-Hb complex prevents filtration of Hb through the glomerulus for renal excretion, and is instead taken to the liver to be broken down into Fe and bilirubin

3. When excess Hb in plasma is present, the Hpt is saturated and unbound Hb is filtered by the kidneys

4. Most unbound Hb is reabsorbed by tubular epithelial cells to be converted to haemosiderin

5. When the capacity for tubular resorption of Hb is exceeded, free Hb is present in the urine

Iron Metabolism

• 3 Storage sites

• Normal metabolism

• Effects of anaemia on iron metabolism

• Transferrin

  • Definition

  • 2 Causes of decreased transferrin concentration

  • 2 Causes of increased transferrin concentration

Storage:

1. Within RBCs (50 - 70%)

2. Within macrophages of bone marrow, spleen and liver (25 - 40%)

3. Small amounts in other molecules (myoglobin and enzymes)

Metabolism: Very little iron is lost

• Iron released from RBC breakdown is almost enough for normal erythropoiesis + small contribution from absorption from gut

Anaemia: Increases iron demand due to accelerated erythropoiesis

1. Increased efficiency of intestinal Fe absorption

2. Release iron from tissue macrophages within haemosiderin or ferritin

Transferrin:

• Definition: Apotransferrin that transports iron in the blood → Used to assess iron levels in blood

• Decreased [transferrin]

  1. Iron deficiency

  2. Acute/chronic inflammation

• Increased [transferrin]

  1. Haemolytic anaemia

  2. Hyperadrenocorticism

TWO Methods of CBC Analysis

• Advantages

• Disadvantages

• Information gathered

ONE: Automated analyser (commercial or in-clinic labs)

+ve: Good at counting

-ve: Unreliable at characterising

Information

1. Erythrogram: Count, Hb, HCT, MCH, MCHC, retics

2. Leukogram: Count and differential BUT not reliable

3. Thrombogram: Count

TWO: Blood smear exam

+ve: ESSENTIAL part of haematological exam

1. Necessary for characterising morphology (eg. inclusions, change in shape etc.)

2. Check if the automated analyser was accurate

Area: Monolayer = Transition between the body and feathered edge of the blood smear (blood cells JUST separated)

Information:

• RBC morphology: Size, colour, shape, other (structure and arrangement)

  • Check the automated analyser counted correctly

• WBC morphology: Differential and stages

• PLT number (clumping)

Describe EIGHT assessments of erythrocytes in the CBC

1. Haematocrit (HCT) = Volume of erythrocytes that make up a L volume of blood

   • Calculated value (HCT = MCV x RBC)

   • Unit: L/L (proportion between 0 and 1)

2. Packed cell volume (PCV) = % of blood composed of erythrocytes

   • Manually measured estimated of HCT by centrifuge

   • Unit: %

3. Red cell count (RBCs) = Number of erythrocytes per unit volume of blood

   • Unit: # erythrocytes x 10^12/L

   • Necessary for MCV and MCH calculations

4. Haemoglobin concentration (Hb) = Amount of Hb per unit volume of blood (lyse RBCs in sample to calculate)

   • Unit: g/L

   • Most direct indication of O2 carrying capacity of blood

5. Absolute reticulocytes (absolute retics) = Number of reticulocytes per unit volume of blood

   • Absolute superior: In anaemic animals, % does NOT account for relative increase in reticulocytes due to the reduced numbers of mature erythrocytes

6. Mean corpuscular volume (MCV) = Volume of an average RBC (size)

   • Unit: fL (femolitre)

   • Helps to classify anaemias

7. Mean corpuscular haemoglobin concentration (MCHC) = Amount of Hb in a litre of RBC (vs. Hb concentration = amount of Hb in whole blood including plasma)

   • Unit: g/L

   • Influences the colour and pallor of the RBC MCHC = Hb/HCT = MHC/MCV

8. Mean corpuscular haemoglobin (MCH) = Amount of Hb per average RBC

   • Unit: Picogram (pg)

   • Little information provided

   • MCH = Hb/RBC

Mean Corpuscular Volume (MCV)

• 4 DDx for macrocytosis

• 3 DDx for microcytosis

Macrocytosis:

1. Regenerative anaemia = Reticulocytosis

2. IMHA (artifactual increase in MCV and decreased RBC count as agglutinated cells are counted as one)

3. Poodles

4. FeLV

Microcytosis:

1. Iron deficiency anaemia (less Fe to synthesise Hb = smaller cell as RBC adjusts to Hb content)

2. PSS

3. Japanese Akitas and Shibas

Structure of a reticulocyte

• Anucleated

• Slightly larger than mature erythrocyte

• Still with some cytoplasmic organelles (mitochondria and ribosomes) = slightly basophilic

• Hypochromic (pallor) due to fewer Hb

Mean Corpuscular Hb Concentration (MCHC)

• 2 DDx for hypochromasia

• DDx for hyperchromasia (+ 6 examples)

Hypochromasia: Dogs = Bigger central pallor and narrow rim of Hb

1. Iron deficiency (lack of Fe = lack of Hb)

2. Regenerative anaemia = Reticulocytosis (immature RBCs have fewer Hb)

Hyperchromasia: Artifactual (each RBC has finite Hb carrying capacity)

1. Haemolysis (extracellular Hb)

2. Heinz bodies

3. Marked spherocytosis

4. Lipaemia

5. RBC shrinkage due to EDTA

6. Crenation

Advia scattergram

Graph produced by the in-lab CBC machines where each dot represents an RBC

Describe SIX features to assess with RBC on blood smear (+ examples)

ONE: NUMBER

Severe anaemia = More gaps between RBCs

• Cannot accurately estimate number from blood smear alone

TWO: SIZE

Anisocytosis = Variation in cell size (large/small/both)

• Indicative of a regenerative anaemia (larger reticulocytes) BUT mild anisocytosis is normal in cats

THREE: COLOUR

Polychromasia = Variation in cell colour and staining affinity due to:

1. [Hb] which affects redness and pallor (increases with maturity)

2. RNA produces blue (ribosomes and mitochondria decrease with maturity)

FOUR: SHAPE

Poikilocytosis = Non-specific abnormally shaped erythrocyte

1. Acanthocyte

2. Echinocyte

3. Schistocyte

4. Keratocyte

5. Spherocyte

6. Eccentrocyte

7. Codocyte/leptocyte

FIVE: OTHER STRUCTURES

1. Basophilic stippling

2. Heinz bodies

3. Howell-Jolly bodies

4. Metarubricytes

5. Siderocytes

SIX: ARRANGEMENT IN MONOLAYER

1. Agglutination

2. Rouleaux formation

Hallmarks of regenerative anaemia

• 3 Hallmarks on CBC

• 3 Hallmarks on blood smear

CBC:

1. Increased reticulocyte count

2. High MCV = Macrocytosis

3. Low MCHC = Hypochromasia

Blood Smear:

1. Anisocytosis

2. Polychromasia

3. ± Basophilic stippling in large animals

TWO methods of reticulocyte staining

• Appearance

• Which can ID punctated reticulocytes?

1. Diff-Quick routine blood staining

   • Polychromatophil = Reticulocyte that is larger and more basophilic due to more ribosomes in the cytoplasm

2. New methylene blue stain (NMB)

   • Stains RNA of immature erythrocytes blue → Blue dots on reticulocytes (can also identify punctated reticulocytes)

     • Aggregated = More organelles in cytoplasm and larger cells that can be identified on Diff-Quick stain (and NMB)

     • Punctated = More mature erythrocytes which have fewer smaller organelles and can ONLY be identified on NMB stain (indicate more chronic regenerative anaemia)

Name the cell

• Definition/appearance

• 2 DDx

Codocyte/Leptocyte

Definition: Excessive membrane resulting in membrane folding and a ring of pallor

DDx: Little significance unless seen in almost every cell

1. 99% artifactual due to increased EDTA

2. Hepatic or lipid disorders

Name the cell

• Definition/appearance

• DDx

Eccentrocyte

Definition: Clear area to the side of the cell and lack of central pallor as Hb shifted to one side

DDx: Oxidative damage of Hb (commonly seen with Heinz bodies)

Name the cell

• Definition/appearance

• DDx

• Location

Spherocyte

Definition: Small, darkly staining, spherical erythrocytes which lack central pallor caused by partial phagocytosis my macrophages in spleen and liver

• Membrane of the RBC close the phagocytosed gap and it becomes smaller

DDx: IMHA

• Easier to detect in dogs as they have a normal centre of pallor

Location: ID in monolayer

Name the cell

• Definition/appearance

• 2 DDx

Keratocyte

Definition: Blister-like vesicle which can rupture → 2 long horns on one side of the cell

• aka. Blister cell, helmet cell, apple stem cell

DDx:

1. Iron deficiency

2. Intravascular trauma (DIC, non-laminar flow or vascular neoplasia)

Name the cell

• Definition/appearance

• 4 DDx

Schistocyte

Definition: Erythrocyte fragments formed from intravascular trauma → Shearing of RBCs

DDx:

1. DIC (fibrin clots shear RBCs)

2. Vascular neoplasia (eg. HSA)

3. Non-laminar flow (eg. valvular disease)

4. Iron deficiency (fragmentation of weaker cells exposed to NORMAL forces)

Name the cell

• Definition/appearance

• 7 DDx

Echinocyte

Definition: Multiple, short, evenly-spaced, uniform projections

• aka. Burr cell

DDx:

1. Artifact #1 (slow drying of blood smear, underfilling EDTA tubes, improper smearing, crenation DDx)

2. Common in cats

3. Renal failure

4. Lymphoma

5. Chemotherapy

6. Rattle snake bites

7. Post-exercise in horse

Name the cell

• Definition/appearance

• 3 DDx

Acanthocyte

Definition: Cell with few, unevenly-spaced, irregular projections due to change in cholesterol/phospholipid concentrations in RBC membrane

• aka. Spur cell

DDx:

1. Dog with splenic haemangiosarcoma

2. Dog with iron deficiency

3. Cat with hepatic lipidosis

What is this?

• Definition

• 2 DDx

Basophilic Stippling

Definition: RBCs with blue dots (ribosome aggregations) in cytoplasm

DDx:

1. Regenerative anaemia (reticulocytosis)

2. Lead poisoning

What is this?

• Definition

• 3 DDx

• Alternative stain

Heinz Bodies

Definition: Unstained blobs at the edge of RBCs on normal stain (blue on NMB stain)

DDx: Oxidative damage → Hb denaturation on the internal RBC surface

1. Oxidative chemicals (brassicas, paracetamol and onions)

2. Most normal cats have <10% RBCs with Heinz bodies

3. >10% = diabetes mellitus, hyperthyroidism and lymphoma

Stain: NMB stain = Blue Heinz bodies

What is this?

• Definition

• 3 DDx

Howell Jolly Bodies

Definition: Small (1µm), purple, spherical inclusion within erythrocytes = Retained nuclear remnants

DDx:

1. Regenerative anaemia

2. Chronic steroid therapy in dogs

3. Reduced splenic function

What is this?

• Definition

• 5 DDx

Metarubricytes

Definition: Last nucleated stage of erythrocyte

DDx:

1. Severe regenerative anaemia

2. Lead toxicity

3. Non-functioning spleen

4. Haemangiosarcoma

5. Increased corticosteroids

What is this?

• Appearance

• 2 DDx

• Alternative stain

Siderocytes

Definition: Mature erythrocytes with iron-containing (siderotic) cytoplasmic inclusions

DDx:

1. Taranaki anaemia (decreased Mg in cattle)

2. Lead toxicity

Stain: Iron granules stain positive with Prussian blue

TWO Abnormal Arrangements of RBCs in the Monolayer

• Appearance

• DDx

• How to differentiate

AGGLUTINATION

Appearance: RBCs grouped in irregular clumps (grapes)

DDx: Ag-Ab complexes (eg. IMHA)

MUST ID in monolayer (thicker layer causes cells to appear clumped)

ROULEAUX FORMATION

Appearance: Linear stacking of cells

DDx: Increased plasma protein content

1. Hyperfibrinogenaemia (acute inflammation)

2. Hyperglobulinaemia (plasma cell myeloma)

3. NORMAL in horses

Differentiate: Saline dispersion test (rouleaux disperse)

Bone marrow examination

• Function

• Always send what?

• 6 Indications

Function: Confirm or exclude DDx based on haematology (when no definitive dx is reached on haematology)

Send: Always sent peripheral blood smear for CBC at the same time

Indications:

1. Non-regenerative anaemia

2. Persistent leukopenia

3. Persistent thrombocytopenia (with no obvious cause)

4. Atypical cells suggestive of leukaemia in peripheral blood

5. Monoclonal gammopathies

6. Determine regenerative anaemia in horses

Polycythaemia

Increase in ALL cells in the blood

Erythrocytosis

• Definition

• 4 Causes

  • Definition

  • Clues

  • DDx

Definition: Increased erythrocyte concentration in blood (PCV, RBC and [Hb] above RR)

Causes:

1. Greyhound

2. Relative Erythrocytosis = Erythrocyte mass is SAME, but plasma volume is reduced

   • Clues: Increased TP and dehydration (skin tent and enophthalmos)

   • DDx:

     1. Dehydration #1 (eg. vomiting/diarrhoea, diuresis, reduced fluid intake)

     2. Endotoxaemia (net movement of water out of vessels via widespread vasodilation)

3. Physiological Erythrocytosis = Transient (<2hr) increased erythrocyte mass due to increased adrenaline (SNS) → Smooth muscle contraction of the spleen → RBCs released into circulation

   • NO pathological significance

   • Clues: Fear, pain, excitement (NOT stress) eg. young horse #1

     • NORMAL TP

     • ± Concurrent physiological neutrophilia

4. Absolute Erythrocytosis = True increase in erythrocyte mass (longer term change)

   1. Primary Erythrocytosis = Increased erythrocyte produced independent of EPO (normal PO2)

      • Rare condition due to myeloproliferative neoplasia (bone marrow) resulting in uncontrolled division of RBCs without EPO via clonal expansion = Polycythaemia vera

   2. Secondary Erythrocytosis = Increased erythrocyte production in response to increased EPO levels

      • Appropriate = Increased EPO associated with tissue hypoxia which is detected by renal interstitial cells and stimulates produced of EPO

        1. High altitude

        2. Chronic respiratory disease causing poor oxygenation of blood

        3. Cardiovascular shunts

      • Inappropriate = Increased EPO WITHOUT hypoxia (normal PO2)

        • DDx: EPO-secreting renal tumours or lesions (renal cysts, hydronephrosis, infarcts)

Overview of Anaemia

• Definition

• 5 Clinical signs

• 3 Mechanisms

Definition: Reduction in RBC mass resulting in reduced RBC count, PCV/HCT and usually [Hb]

Clinical Signs:

1. Pale MM

2. Lethargy

3. Reduced exercise tolerance

4. Increased RR

5. Increased HR ± murmurs due to turbulence

Mechanisms:

1. Decreased RBC production

2. Increased RBC destruction (haemolysis)

3. Increased RBC loss (haemorrhage) COMBINATIONS of them too

Fill out the following table

Classification of Anaemia	DDx
Normocytic, normochromic
Macrocytic, hypochromic
Microcytic, hypochromic
Microcytic, normochromic

Classification of Anaemia	DDx
Normocytic, normochromic	Non-regenerative anaemia
Macrocytic, hypochromic	Regenerative anaemia (polychromatophil)
Microcytic, hypochromic	Iron deficiency anaemia (lack of Hb)
Microcytic, normochromic	Early iron deficiency anaemia Hepatic disease/portosystemic shunts

List 7 other tests to help diagnose the cause of anaemia

1. Estimation of plasma protein = Indicates regenerative anaemia due to blood loss (low) OR haemolysis (normal)

2. Coombs test for suspected immune-mediated haemolytic anaemia

3. Microcytic anaemia → Check faeces for blood (chronic blood loss from GIT disease causing iron deficiency anaemia)

4. Serum iron concentrations

5. Ehrlichiosis check for dogs with thrombocytopenia and hyperglobulinaemia (not in NZ)

6. FeLV and FIV test for anaemic cats

7. EIV test for anaemic horses

Describe the THREE types of anaemia

• Definition

• Timing

• Causes

ONE: REGENERATIVE ANAEMIA

Definition: Bone marrow response to anaemia by increasing synthesis of EPO which accelerating erythropoiesis and release of immature RBCs into circulation

• Shorter lifespan of metarubricytes and reticulocytes and poorer O2 carrying capacity

Timing: First visible 3 - 5 days loss and peak 7 days

Causes:

1. Haemorrhage (RBC lost as whole blood)

   1. Acute vs. chronic blood loss

   2. Internal vs. external blood loss

2. Haemolysis (RBC ONLY destruction)

   1. Intravascular

   2. Extravascular

TWO: PRE-REGENERATIVE ANAEMIA

Definition: Not enough time elapsed for evidence of bone marrow regenerative response to be seen in the peripheral blood smear

Timing: Sample taken within 3 - 5 days post-loss

Diagnosis: Differentiate by repeating the CBC 3 - 5 days later to observe for change (i.e. reticulocytes present)

NON-REGNERATIVE ANAEMIA

Definition: Lack of expected bone marrow response (bone marrow cannot produce enough RBCs to replace those lost normally)

Timing: Diagnose with at least 2 CBCs to assess change in reticulocyte count (stays low = non-regenerative, increases = pre-regenerative → regenerative)

Causes:

1. Deficiency of essential erythropoietic factor

2. Toxins inhibiting bone marrow function

3. Infection

4. Neoplasia

5. Defective maturation of RBCs

6. Deficiency in stem cells

Timeframe of acute blood loss

Time Post-Haemorrhage	RBC Parameters	Other Signs
<4hr
4 - 12hr
3 - 5d
7d
10 - 14d

Time Post-Haemorrhage	RBC Parameters	Other Signs
<4hr	NORMAL ± Increase in PCV due to splenic contraction	± PLT increase and mild neutrophilia by 3hr
4 - 12hr	Reduced PCV Haemodilution = Blood volume restoration from interstitial fluid	Reduced TP
3 - 5d	Reticulocytosis
7d	Peak reticulocytosis	Normal TP
10 - 14d	Normal	Normal

Chronic Haemorrhage

• Definition

• Clinical signs

• RBC changes

• 3 DDx

Definition: Anaemia develops slowly without hypovolaemic shock and provides the body time to adapt

Clinical Signs: May develop AFTER drop in PCV

• Regenerative response is evident initially with low TP, but it becomes progressively less regenerative overtime due to iron deficiency

RBC: Iron deficiency anaemia = Microcytosis and hypochromasia

DDx:

1. Internal parasitism (eg. haemonchosis, hookworms, coccidiosis)

2. External parasitism (eg. fleas, ticks, lice)

3. GIT ulcers

Internal Haemorrhage vs. External Haemorrhage

• Definition

• Process and regenerative response

• Causes

Internal Haemorrhage

Definition: Haemorrhage into tissues or a body cavity

Process: Most RBCs resorbed by lymphatics and recirculate within 24 - 72hr

• Remaining RBCs are phagocytosed and lysed with the iron and plasma proteins reutilised → Greater regenerative response

• Difficult to differentiate from haemolysis due to normal TP (rapid resorption via lymphatics)

Causes:

1. Bleeding in lungs, liver, within body cavities

2. Bleeding disorders (eg. haemophilia, thrombocytopenia)

3. Bleeding tumours

External Haemorrhage

Definition: Whole blood loss from body (RBC, plasma protein, iron)

Process: Decreased TP with reduced regenerative response (plasma proteins and iron lost)

Causes: Haemorrhage from

1. GIT

2. Respiratory tract

3. Skin

4. Urinary tract

Overview of Haemolytic Anaemia

• Definition

• 2 Signs/clues

• 2 Types

Definition: Accelerated RBC destruction which cannot be balanced by increased erythropoiesis

Signs:

1. Normal TP

2. Larger regenerative anaemia and more reticulocytes due to recycling of iron (retained in plasma)

Types:

1. Intravascular haemolysis

2. Extravascular haemolysis

9 DDx for regenerative anaemia and 5 DDx for non-regenerative anaemia

Regenerative Anaemia:

1. IMHA (5)

2. Neonatal isoerythrolysis (3)

3. Iron deficiency anaemia (4)

4. Sporidesmin toxicity (3)

5. Leptospirosis (3)

6. Chronic copper deficiency (3)

7. Blood parasites (3)

8. Brassica redwater (2)

9. Bovine post-parturient haemoglobinuria (2)

Non-Regenerative Anaemia:

1. Anaemia of chronic inflammatory disease

2. Anaemia of chronic renal failure

3. Iron deficiency anaemia (4)

4. Lead toxicity (4)

5. “Taranaki” Anaemia (2)

Immune-Mediated Haemolytic Anaemia (IMHA) (5)

• Primary vs. secondary

• Pathogenesis

• Species variation

  • Dog (aetiology and signalment)

  • Cat (aetiology and signalment)

  • Horse (aetiology and signalment)

• 6 Clinical features

• 6 Laboratory findings

• Diagnosis (+ 3 -ve)

Primary vs. Secondary:

1. Primary IMHA = Autoimmune in which the body attacks own RBC Ag

2. Secondary IMHA = Antigenic alteration in RBC due to infectious agents, inflammatory agents, neoplasia or drugs

Pathogenesis:

1. Ab developed against Ag on the RBC surface with complement fixation

2. Recognised by macrophages in the spleen and bone marrow

3. Extravascular haemolysis which develops to intravascular (specific diseases may directly be intravascular)

Species Variation: Dogs > Cats » Horses

• Dogs = Primary #1

  • Females > Males

  • Cocker spaniel, standard poodle, rough coated collie, old English sheepdog

• Cats = Secondary #1 (Mycoplasma haemofelis, FeLV or neoplasia)

  • Worse prognosis than dogs (cannot be treated with corticosteroids)

• Horses = Secondary and RARE

Clinical Features: Regenerative extravascular ± intravascular haemolytic anaemia

1. General anaemic signs

2. Haemoglobinaemia

3. Icterus

4. Splenomegaly (more splenic macrophages)

5. ± Hepatomegaly (icterus-related)

6. ± Fever (infectious agent or neoplasia)

Laboratory Findings:

1. Moderate - severe anaemia (low HCT, Hb, RBC)

2. Marked regenerative response (anisocytosis, polychromasia and reticulocytes ± nRBC)

3. SPHEROCYTES and ghost cells (no Hb)

4. Agglutination which is grossly visible if severe

5. ± Leukocytosis with neutrophilia and left shift

6. ± Thrombocytopenia

Diagnosis: Positive Coomb’s test = Detect anti-RBC Ab (reagent to species-specific anti-Ig and/or C3b)

-ve:

1. Helps confirm IMHA but has poor Sn and Sp

2. NOT necessary if gross agglutination is observed on smear

3. Does NOT work for other suspected immune-mediated diseases

Neonatal Isoerythrolysis (3)

• Species signalment

• Pathogenesis

• Diagnosis

• Treatment

Species: Foals #1

Pathogenesis: Regenerative extravascular haemolytic anaemia

1. Foal inherits RBC Ag from stallion which are incompatible with the mare

2. Small amounts of the foetal RBC Ag enter the maternal circulation across the placenta due to haemorrhage

3. Maternal Ab raised to foal RBC Ag and concentrate in mare's colostrum

4. Foal is normal at birth, but develops clinical signs within 24 - 48hr of colostrum intake

   • RARE in maiden mares as the first foal is the exposure (insufficient time for adaptive immune response)

Diagnosis: Test mare for anti-RBC Ab against stallion's RBCs 2 - 3 weeks prior to foaling Coomb's OR haemolytic tests

Treatment: Withhold mares colostrum in first 24 - 36hr and feed substitute

• After delay, foal can feed safely as not enough Ab absorbed to cause haemolysis

Iron Deficiency Anaemia (4)

• 3 DDx

• Features on blood smear

• Diagnosis

DDx: Fairly common

1. Chronic blood loss (esp. GIT) #1

2. Suckling animals (piglets in commercial operations) which have no soil and milk is low in Fe

3. Puppies and kittens heavily infected with fleas and hookworms

Blood Smear: Regenerative → Non-regenerative as iron in tissues is depleted

1. Microcytic ± hypochromic

2. RBC fragility = Schistocytes keratocytes, blister cells and apple stem cells

3. Thrombocytosis in 50% of cases

Diagnosis:

Tests for transferrin saturation (low indicates iron deficiency anaemia)

Blood Parasites (3)

• 2 MoA causing haemolysis

• Risk factor

• Agents (cat, sheep, cow, pig, dog)

• 2 Methods of diagnosis

MoA:

1. Immune-mediated extravascular haemolysis (parasites bind outside of RBC and Ab bind to increase macrophage phagocytosis)

2. Intravascular haemolysis (direct eg. Babesia)

Risk: ONLY causes disease in immunosuppressed individuals or those with concurrent disease (incidental findings in NZ)

Agents:

• Cat = 3 types of mycoplasma (M. haemofelis most pathogenic)

  • Small purple dots on outside of RBC surface

  • Take blood smear IMMEDIATELY after sampling as parasites fall off

• Sheep = Mycoplasma ovis in NZ but no clinical significance

• Cow = Mycoplasma wenyoni and Theileria orientalis

• Pig = Mycoplasma haemosuis

• Dog = Babesia spp. and Anaplasma spp. (EXOTIC)

  • Tick-borne parasites (competent vectors in NZ)

Diagnosis:

1. Blood smear

2. PCR assay which have higher specificity and sensitivity

Brassica Redwater (2)

• Signalment

• Aetiology

• Clinical signs

Bovine Post-Parturient Haemoglobinuria (2)

• Signalment

• Aetiology

• Clinical signs

Brassica Redwater

Signalment: Ruminants

Aetiology: Brassica crops (kale/rape and Field Mustard)

• Unpredictable and transient toxicity causing oxidative damage

Clinical Signs: Regenerative intravascular haemolytic anaemia

• Heinz bodies

Bovine Post-Parturient Haemoglobinuria

Signalment: Sporadic disease of multiparous, high-producing dairy cows (within 4 weeks of calving)

Aetiology: Phosphorus deficiency or Northland cattle

Clinical Signs: Regenerative intravascular haemolytic anaemia

10 Toxic causes of oxidant-induced haemolytic anaemia

1. Onion (dog and cat)

2. Brassica (contain SMCO)

3. Wilted red maple leaves (camelid and horse)

4. Pistacia leaves (horse)

5. Nitrates (ruminants)

6. Zinc (dog)

7. Copper

8. Moth balls (dog)

9. Skunk musk (dog)

10. Paracetamol (cat)

Non-Regenerative Anaemia

• Definition

• Classification

Definition: Associated with bone marrow that cannot produce enough RBCs ± other cell lines involved

Classification: Whether or not other cell lines are affected too

1. Pancytopenia due to aplastic anaemia

2. RBC line only

   1. Pure red cell aplasia

   2. Red cell hypoplasia

Aplastic Anaemia

• Definition

• 2 Causes

Definition: Decrease in ALL cell lines (red, white and platelets) = Pancytopenia

Causes:

1. Damage to stem cells (drugs, hormones or toxins)

   • eg. Chemotherapy or lead toxicity

2. Myelophthisic anaemia (replacement of functional haematopoietic tissue with another tissue eg. neoplasia, fibrous tissue, bone = osteopetrosis)

Pure Red Cell Aplasia

• Definition

• Incidence

• Example

Definition: Increased destruction of RBC line

Prevalence: RARE intrinsic bone marrow problem

Example: IMHA of erythroid precursors in dogs

Red Cell Hypoplasia

• Definition

• 4 Examples

  • MoA

  • DDx

Definition: Externally-originating bone marrow condition resulting in decreased erythropoiesis

Examples:

1. Anaemia of chronic disease #1

   • MoA:

     1. Inflammatory cytokines suppress erythropoiesis (IL-1, TNF-⍺, IFN-ɣ)

     2. Iron sequestration

     3. Reduced RBC lifespan

   • DDx: Chronic disease due to inflammation, infection, trauma or neoplasia

2. Anaemia of chronic renal disease

   • MoA: Decreased EPO (also other causes like chronic inflammatory disease mechanisms)

   • DDx: Chronic renal failure

3. Anaemia of endocrine disease

   • DDx: Hyperthyroidism and hypoadrenocorticism

4. Anaemia of nutritional deficiency

   • DDx: Iron deficiency (chronic loss NOT inadequate intake), copper, vitB6, cobalamin, cobalt deficiency

Lead Toxicity (4)

• Signalment

• Pathogenesis

• 2 Features on blood smear

Signalment: Young dogs

Pathogenesis: Lead inhibits breakdown of ribosomal RNA and heme synthesis

Blood Smear: Mild non-regenerative (or poorly regenerative) anaemia

1. Inappropriately high metarubricytes and polychromatic cells for mild anaemia

2. Basophilic stippling of non-cattle RBCs esp. in metarubricytes

Taranaki Anaemia (2)

• Species signalment

• Aetiology

• 2 Clinical signs

• 2 Features on blood smear

Signalment: Rare disease in Taranaki dairy cows ± chronic udder oedema

Aetiology: Long-term magnesium deficiency

Clinical Signs:

1. Poor BCS with low milk yield

2. Mild non-regenerative anaemia

Blood Smear:

1. Moderate - severe metarubricytes

2. Variable siderocytes

EXAMPLE: Greyhound with poor performance

Question: Provide description and interpretation for the case

Description

Mild erythrocytosis (high Hb, PCV, RBC)

TP - unremarkable

Interpretation

Given that the total protein did not reveal any significant findings (eg. high TP indicative of dehydration), and Greyhounds normally have a high PCV, this animal is normal. The reference ranges provided are not specific for greyhounds.

EXAMPLE: 8-month-old Friesian calf with marked exercise intolerance and rapid RR. MM cyanotic and heart sound abnormal.

Question: Provide an interpretation and diagnosis for the case

Interpretation: Marked erythrocytosis (high Hb and PCV) likely due to inadequate oxygenation of blood (indicated by the exercise intolerance, rapid RR and cyanosis) caused by a cardiovascular shunt (consistent with the heart murmur). More indicative of absolute erythrocytosis due to marked increase, and there is normal protein (less likely to be relative). Physiological is less likely as it is a cow. The cardiovascular shunt would prevent Hb from picking up O2 in the lungs, resulting in more deoxygenate Hb in the blood which would cause the cyanotic membranes. The kidneys detect hypoxia and secrete EPO in response to increase erythropoiesis in the bone marrow to release more RBCs necessary to increase O2 carry capacity of blood.

Mild leukocytosis (increased total WBCs) characterised by moderate mature neutrophilia and monocytosis. Indicative of inflammatory disease that is chronic in ruminants (also consistent with normal fibrinogen which means it is less likely to be acute), likely due to the hypoxia of the tissues. Also indicative of a stress leukogram and a result of chronic disease causing increased production in corticosteroids.

Diagnosis: Cardiovascular shunt (heart disease)

Classification: Absolute secondary appropriate erythrocytosis

EXAMPLE: Cat HBC and immediately brought into vet. MM pale and owner reports significant blood loss

Question: Provide an interpretation and diagnosis

Interpretation: All parameters within normal reference ranges as cat was brought into clinic immediate after haemorrhage occurred. Not enough time has elapsed to observe any changes in blood as this was haemorrhage and blood was lost in equal proportions of blood cells to plasma.

Diagnosis: External haemorrhage <4hr ago (cat will become anaemic but is not apparent yet from CBC)

EXAMPLE: 3-year-old Friesian been off milk for 4 days and depressed

Question: Provide and interpretation and diagnosis

Interpretation: Moderate anisocytotic, hyperchromic (high MCHC) regenerative (high reticulocytes, basophilic stippling and nRBC present) anaemia (low Hb and PCV). Hyperchromasia likely artifactual due to EDTA artefact or haemolysis within the tube (RBC cannot be more hyperchromic as finite room for Hb).

This regenerative anaemia may be due to blood loss or destruction. I think we are leading towards a blood loss due to the marked panhypoproteinaemia (low TP) characterised by moderate hypoalbuminaemia and hypoglobulinaemia (low Albumin and Globulin) as RBC destruction does NOT change hypoproteinaemia. This low protein also is indicative of external haemorrhage as is is a marked decrease (internal haemorrhage: protein can be resorbed into circulation).

Mild mature neutrophilia (increased segmented neutrophils) likely due to corticosteroid response due to being unwell. It is unlikely to be physiologic as it is an adult cow. It could be mild inflammation, but the normal fibrinogen means this is less likely. Monocytopenia is unlikely clinically relevant.

Diagnosis: Regenerative anaemia due to external blood loss (abomasal ulceration is a common cause)

Classification: Moderate regenerative anaemia likely due to external haemorrhage

EXAMPLE: 2-year-old Labrador presented for vomiting and seizures

Question: Provide an interpretation and diagnosis

Erythrocyte changes: Moderate, normocytic (normal MCV), normochromic (normal MCHC), pre-regenerative or non-regenerative (normal absolute retics.) anaemia (low Hb, PCV, RBC) with high nucleated RBC and moderate basophilic stippling.

Leukocyte changes: None

Diagnosis: Lead toxicity. Basophilic stippling in non-ruminant species is highly indicative of lead toxicity as is the inappropriately high number of metarubricytes for mild - moderate anaemia observed.

Classification: Mild to moderate, normocytic, normochromic, non-regenerative anaemia