Week 9: Blood Examination & Properties

Lecture part 1 + 2. finished.

BLOOD PROPERTIES

• Volume: 6-8% of body weight

• pH: Approximately 7.4

• Color:

  • Arterial blood: Red

  • Venous blood: Dark red

• Odor: Specific

• Taste: Slightly salty

Functions of Blood 🩸

Blood serves three primary functions:

1. Supply:

   • Oxygen

   • Essential nutrients

   • Enzymes

   • Hormones

   • Water and electrolytes

   • Buffering system to maintain constant conditions

2. Removal: Metabolic waste products

3. Defense:

   • Blood cells (leukocytes)

   • Blood proteins (immunoglobulins - antibodies)

Blood Composition 🧬

Blood consists of two main components:

• Plasma: Contains fibrinogen

• Blood corpuscles

Blood Cells (Static) Forming Organs 🩸

• Hematopoiesis: The process of blood cell formation and development.

• All blood cells originate from the stem cell called the hemocytoblast.

• Hemocytoblast → Erythroid stem cell → RBC (if becoming a red blood cell)

• Forming organs:

  • Bone marrow

  • Liver

  • Spleen

  • Lymph nodes

  • Reticulo-endothelial tissue

Functions of the Spleen 🫘

• Blood reservoir: Storage site for red blood cells and platelets; the body can contract the spleen to release more red blood cells.

• Site of immune activities: Part of the immune system.

• Blood filtration: Removes old or abnormal blood cells and particles.

• Hematopoiesis: Produces certain blood cells and is a major site for red blood cell production outside the bone marrow.

• Phagocytosis: Traps and removes old cells, bacteria, and foreign proteins from circulation.

Haematology 🧪

Study of blood and blood-forming organs.

Haematological Parameters

• Red Blood Cell count (RBC): [Tera (10^12)/l]

• White Blood Cell count (WBC): [Giga (10^9)/l]

• Packed Cell Volume (PCV): [% or l/l]

• Haematocrit (HCt): [l/l]

• Haemoglobin (Hb): [g/l or g/dl]

• Mean Cell Volume (MCV): [fl]

• Mean Corpuscular Haemoglobin (MCH): [pg]

• Mean Corpuscular Haemoglobin Concentration (MCHC): [mmol/l or g/dl]

• Platelets count: [Giga (10^9)/l]

• Differential WBC: (lymphocytes, monocytes, neutrophils, eosinophils, basophils) [% and Giga (10^9)/l]

• Reticulocytes count: [‰]

Haematological Examination Methods

1. Routine methods

2. Electrical impedance methods

3. Flow cytometry methods

Blood Sampling Conditions 🩸

• Avoid excitement

• Disinfect the venipuncture site

• Use dry needles (to prevent hemolysis)

• Limit pressure for vein distension to < 2 minutes (to avoid haemoconcentration, hemolysis)

Blood Sampling Sites by Animal 📍

• Cattle: Jugular vein, subcutaneous abdominal vein (anterior mammary), middle coccygeal vein

• Small Ruminants: Jugular vein

• Horse: Jugular vein

• Pigs:

  • Small pigs: V-shaped trough

  • Large pigs: Loop noose around the snout

  • Ophthalmic (orbital sinus)

  • Puncture of ear vein with surgical blade or needle (for small blood quantities)

  • Amputation of the tail tip

  • Anterior vena cava

• Dog and Cat: Cephalic (radial) vein, recurrent tarsal (saphenous) vein, jugular vein

• Lab animals (rabbit, guinea pig, rats): cardiac puncture

Blood Quantity 🩸

• Normovolaemia: Normal volume of blood (7-8% of body live weight)

• Hypovolaemia: Haemoconcentration (higher haematocrit). Causes: diarrhea, sweating, bleeding

• Hypervolaemia: Hydraemia (low haematocrit). Causes: liver disease (cirrhosis), kidney failure (loss of erythropoietin), heart insufficiency

Blood Color 🎨

• Normal:

  • Venous: Dark red

  • Arterial: Bright red

• Pathology:

  • Venous blood − pale: cyanide poisoning, leukaemia

  • Increase in CO2 (lungs, heart, bloating,...): dark

  • Nitrites poisoning − cherry-like

  • CO poisoning

Blood Coagulation = Haemostasis 🩸

A process causing bleeding to stop, meaning to keep blood within a damaged blood vessel (the opposite of haemostasis is hemorrhage). It is the first stage of wound healing; this involves blood changing from a liquid to a gel. Haemostasis also helps us to find out why a patient might have a bleeding disorder (hemorrhagic diathesis).

A. Bleeding Time

1. Small puncture wound

2. Swabbing away in 15–20 s intervals

   • Normal time: 3–5 minutes

   • Haemorrhagic diathesis: 10–20 minutes

B. Clotting Time

1. A drop of blood is placed on a transparent base (microscope or watch glass)

2. Every 30 s, a fine needle is inserted into the drop until threads of fibre begin to adhere

   • Normal time: 2–5 minutes

Erythrocyte Sedimentation Rate (ESR)

• Measures how quickly RBCs settle at the bottom of a test tube

• Normally, RBCs sink slowly, as it weights higher than that of plasma.

• If they sink faster, it indicates inflammation in the body.

  • factors that increase ESR:

    • Low hematocrit and blood viscosity - fewer RBCs

    • high fibrinogen levels - in pregnancy, vascular and heart diseases

    • Macrocytic RBC - larger

    • high WBC count

  • Factors that decrease ESR:

    • high hematocrit - more RBCs

    • change in RBCs shape

    • high albumin levels

Sedimentation Rate ⏳

The Westergren tube is used to measure the sedimentation rate of blood.

Horse: vertical - 5 (3.7), 10 (14.7), 30 (69,3), 24H (126nm).

Anticoagulant Additives

Methods: Haematocrit ⚗

1. Routine Blood Count Methods

• Packed Cell Volume (PCV) – proportion of volume erythrocyte to blood

  1. MACRO (Wintrobe tubes)

     • Centrifugation for 30 min at 3000 rpm

  2. MICRO (capillary tubes)

     • Centrifugation for 5 min at 5000 rpm

• The blood sample should be continuously gently shaken every time before examination.

• Macrohematocrit – Wintrobe tubes

Normal Range & Pathological Changes

haemoconcentration - Increased PCV - dehydration, decreased water intake, decreased food intake.

Hydraemia - decreased PCV - bleeding, chronic disease (parasites), decreased erythrocyte size.

Haemoglobin 🩸

Methods

1. Sahli method

2. Colorimetric method (haemolysis)

Normal Range

Pathological Changes

• anemia

• haemoglobinemia (free Hb in blood)

  • hemolysis, water poisoning, blood parasites, beet sugar leaves poisoning and decrease of P.

    • hemoglobinuria

Erythrocytes = Red Blood Cells 🔴

• Erythropoietin: Inductor of erythropoiesis

• Size: 4 – 8 µm

• Life Span:

  animal	days
  dog	140 – 150
  cat	80 – 120
  horse	68 – 77
  pigs	110 – 120
  cattle	62 – 70
  sheep	52

• Non-mammals: Nucleated and elliptical

• Mammals: Non-nucleated

Red Blood Cell Morphology

Erythrocyte Count

Microscopic Method

• Objective 10

• Hayem's diluting fluid (4975 μ l + 25 μ l blood)

• Thoma blood celles diluting pipettes

• Haemocytometer (Bürker's chamber) rule of the right angle

Normal Range

Erythrocyte Indices 📊

A. MCV (Mean Corpuscular Volume) [fI]

MCV=(PCV×1000)/RBCMCV=(PCV×1000)/RBC

• Use: classification of anaemia

  • Normocytic

  • Microcytic

  • Macrocytic

B. MCH (Mean Corpuscular Haemoglobin) [fmol]

MCH=(Hb×6.207)/RBCMCH=(Hb×6.207)/RBC

• Is the average mass of hemoglobin per red blood cell in a sample of blood

C. MCHC (Mean Corpuscular Haemoglobin Concentration) [mmol.l^-1]

MCHC=(Hb×0.6207)/PCVMCHC=(Hb×0.6207)/PCV

• Use: classification of anemia

  • Normochromic

  • Hypochromic

  • Hyperchromic

Normal Range

Erythrocyte Pathology

A. Shape

• Poikilocytosis: Variation in shape

  • Degeneration

B. Size

• Anisocytosis: Variation in size

  • Common feature in animals

  • Red blood cell distribution width (RDW) [%]

    • measure of variation of RBC volume reported as part of standard complete blood count.

    • RBCs - standard size of 6-8 μm in diameter.

    • Higher RDW: high variation in size - anisocytosis.

C. Number

• Reduction: ANAEMIA

• Polycythaemia:

  • Dehydration

  • Impairment of respiratory efficiency (heart, lungs diseases, erythropoietin)

  • Possible solution = Blood transfusion, which is generally the process of receiving blood products into circulation intravenously. Transfusions are used for various medical conditions to replace lost components of the blood.

Etiological Classification of Anaemia

a) Blood Loss Anaemia

• Bleeding (ulcers, enteritis, coccidiosis, neoplasma haemophilia, vitamin C and K deficiencies)

• Blood sucking parasites (endo, ecto)

• Poisonings (warfarin)

• Trauma, surgery

b) Haemolytic Anaemia

• Infectious – Babesia, Anaplasma, Eperythrozoon (felis, suis), Clostridium haemolyticum

• Toxic – copper, lead, mercury, rape, kale, water, furazolidon, incompatibe blood transfusion, beet sugar leaves

c) Dishaemopoietic Anaemia (Hypoplastic or Aplastic Anaemia)

• Depression of erythrogenesis – anemia resulting from an inability of the bone marrow to produce red blood cells

• Nutritional factors (vitamins B1, B2, B12)

• Parasites

• Hypothyroidism

• Irradiation

• Sulphonamides

d) Anemia of Chronic Disease

e) Hereditary Spherocytosis

f) Certain Hereditary Hemoglobinopathies

• Including some cases of thalassemia minor

Symptoms of Anaemia

• CNS: Fatigue, dizziness, fainting

• Eyes: Yellowing

• Skin: Paleness, coldness, yellowing

• Blood Vessels: Low blood pressure

• Heart: Palpitations, rapid heart rate, chest pain, angina, heart attack

• Respiratory: Shortness of breath

• Muscular: Weakness, recumbency

• Intestinal: Changed stool color

• Spleen: Enlargement

Leukocytes ⚪

Types

A. Polymorphonuclear cells (granulocytes) B. Mononuclear cells (agranulocytes)

Life Span

• Lymphocytes: Few hours to a month

• Granulocytes: Less than 14 days

Granulocytes

1. Neutrophils

2. Eosinophils

3. Basophils

Neutrophils characteristics:

• 10 – 15 µm

• Ragged (mature) or smooth (immature) nuclear membrane

• Acidophilic cytoplasm (light pink)

• Pinkish granules

• Deeply basophilic nucleus

• Neutrophils are more or less regular in size across species, compared to RBC

Eosinophils characteristics:

• 10 – 14 µm

• Pale-grey cytoplasm

• Orange-red granules

• Nucleus frequently non-segmented

Basophils characteristics:

• 12 – 22 µm

• Purplish granules

• Nucleus less intense stained

Lymphocyte Details (agranulocyte)

Lymphocytes can be small, intermediate, or large. In cattle, up to 50% can be intermediate. Here's a breakdown:

• Nt-intermediate: Nucleus size is the same as Nt-large.

• Nt can fit inside the nucleus (5-18 µm).

• Blue narrow cytoplasm.

• Excentric rounded dark blue nucleus (bluish granules – vacuoles).

• Most are smaller than a neutrophil.

• Lymphocytes are small, intermediate, or large. Small and mature lymphocytes are most common in peripheral blood.

• In cattle, lymphocytes are in a 1:1 ratio with neutrophils.

Monocytes (agranulocyte)

• 12–20 µm (largest)

• Variable shape nucleus

• Net-like staining pattern

• Factors produced at sites of inflammation can increase Mo production

White Blood Cell Functions 🔬

Here's a summary of the functions of different types of white blood cells:

1. Neutrophils

   • Phagocytosis of small particles.

   • Cellular defense mechanism (inflammation, bacteria).

2. Eosinophils

   • detoxification (histamine inactivation)

   • anti-parasitic defense mechanism

3. Basophils

   • Heparin and histamine release (increasing capillary permeability).

4. Lymphocytes

   • Cell-mediated immunity.

   • Immunoglobulin production.

5. Monocytes

   • Phagocytosis of large particles.

   • Wandering macrophage (tissues).

In leucocyte count - Giga per liter (G/l)

Total Leukocyte Count 🔢

Microscopic - flask method:

• Türk's diluting fluid (475 μl + 25 μl blood)

• Sample tubes or Thoma blood cells diluting pipettes

• Haemocytometer = Bürker's chamber

Here are the normal ranges for total leukocyte count:

Differential Leucocyte values 🩸

• Obtained from stained blood smears.

• Best smears are made on clean, grease-free, dry glass slides.

  • A small drop of blood is placed near the right-hand end of a horizontally situated glass slide.

  • Another slide called "spreader" (with shortened width by one-third) is held at a 30–45° angle.

  • The angled slide is pushed (spreader) to the left.

  • The dried blood smear is immediately stained with Leukodiff 200.

  • differential WBC count is gotten by counting 100-200 WBC, using the meander method and immersion lens of microscope.

  • Such stained blood film can be examined even for RBC parasites.

  • To obtain the percentage value of each cell type, make a written record.

Here are the normal ranges for differential leukocyte values:

Common Counting Errors ❌

1. Improper lighting of the microscope field

2. Careless examination and enumeration of cells

3. Dirty equipment or diluting fluid

4. Poor staining technique (stain too light or too dark)

5. Improperly prepared blood films (too thick or too thin)

Leucocyte Pathology 🩺

1. Total WBC

   • Leucocytosis: ↑ number of leucocytes

   • Leucopenia: ↓ number of leucocytes (infectious feline enteritis)

2. Differential WBC

   • Neutrophils

     • Neutrophilia

     • Infective inflammatory processes

     • Stress

     • Hereditary defects in calves

     • Neutropenia

       • Earlier phases of viral infection (mouth foot disease, canine distemper, canine inf. hepatitis)

   • Lymphocytes

     • Lymphocytosis

     • Lymphopenia

   • Monocytes

     • Monocytosis

       • Feature of recovery

       • Protozoal infections

       • Chronic infections (brucellosis, Listeria mono.)

   • Eosinophils

     • Eosinophilia

       • Allergic conditions

       • Eosinophilic myositis

       • Parasitic diseases

   • Basophils

     • Basophilia: Parenteral introduction of heterologous

     • Allergic conditions

     • Protein

     • Mycosis

     • Decreased function of the thyroid gland

     • Sideropenic anemia

• Left shift: ↑ immature forms (bad prognosis)

• "Degenerative left shift": ↑ immature forms + total WBC normal or only slightly increased

• Shift to the right: ↑ mature forms (inflammation)

Disease Progression Pattern 🔄

• Early stages

  • Absolute neutrophilia (↑ immature forms)

  • ↓ lymphocytes (often relatively)

  • Transient neutropenia (most marked in viral diseases)

• Disease subsidence

  • ↓ neutrophils

  • ↑ lymphocytes and monocytes

• Recovery

  • ↑ lymphocytes and eosinophils

  • Gradual return to the normal leukocyte picture

Thrombocytes (Platelets) 🩸

• Platelets are produced in blood cell formation (thrombopoiesis) in bone marrow by budding off from megakaryocytes.

• The physiological range for platelets is 150–400 G/l.

• The lifespan of circulating platelets is 5 to 9 days.

• Megakaryocyte and platelet production is regulated by thrombopoietin, a hormone usually produced by the liver and kidneys.

• Old platelets are destroyed by phagocytosis in the spleen and by Kupffer cells in the liver.

• Reserve platelets are stored in the spleen and are released when needed by sympathetically induced splenic contraction.

Electrical Impedance Methods (Blood Cell Analyzers) ⚡

• This method counts blood cells by measuring how they change electrical resistance as they pass through a tiny opening. Cell size matters, as the size determines how the electrical resistance changes, allowing the machine to count and differentiate cells based on size.

• Limited differentiation: due to its reliance only on size, it cannot distinugish between cells of the same size but with different internal structures. For ex. cannot tell difference of reticulocytes and erythrocytes, or band neutrophils from segmented.

• Popular method: despite its limitations, its widely used method in hematology analyzers.

• Only 3 WBC populations are counted: lymphocytes, monocytes and granulocytes.

Flow Cytometry Methods (Blood Cell Analyzers) 🔬

• Considered the best method for the differentiation of cell populations.

• Measures multiple characteristics of individual particles flowing in single file in a stream of fluid.

• Light scattering at different angles can distinguish differences in size and internal complexity.

• Light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens.

• This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time.

• It cannot separate reticulocytes from erythrocytes or band neutrophils from segment neutrophils depending on their internal structure.

• Dot plots are a visual representation of the complete blood count (CBC); each dot represents a single cell. Dot plots are a critical element of the CBC, providing a snapshot of cellular morphology.

Comparison of Blood Count Methods ⚖

1. Electrical Impedance

   • Small amount of blood.

   • Gives quick information (3 min) about blood count.

   • Depends only on the size of cells without their intracellular differentiation.

   • Can mistake small Ec for large Tc or small Lc, which can give incorrect results.

   • Pathological states in the blood picture must be checked by manual blood count.

   • In ruminants, manual differential WBC is necessary.

2. Flow Cytometry

   • Very expensive.

   • Small amount of blood.

   • One of the newest, most correct, and precise methods.

   • Can detect external and intracellular antigens of various characters.

   • Can differentiate cells by their size and granularity.

   • Gives complete differential WBC.

   • Can detect immature cells (band neutrophils and reticulocytes).

3. Routine Blood Count

   • The cheapest.

   • The slowest (45 min).

   • Requires skills and routine.

   • Manual dilution affects precision.

   • Requires a greater amount of blood, smear, and staining.

   • Differential WBC (30 min).

Examples of Changes in Leukograms 🐮

Dairy Cow, 1st Sampling, Diarrhea

• Minimum RBC variation in size and shape.

• Large central pale zone.

• RBC fragments.

• Rouleaux formation.

• Small lymphocytes.

• Segmented and band neutrophils.

• Increased serum protein.

Dairy Cow, 2nd Sampling, Diarrhea

• Varying RBC size.

• Smaller central pale zone.

• RBC fragments.

• Acanthocytes.

• Large and small dotted lymphocyte.

• Hypersegmented large neutrophils.

Dairy Cow, 3rd Sampling, Diarrhea

• Acanthocytes.

• Dotted RBC with changed size.

• Howell-Jolly’s bodies.

• Missing central zone.

• Segmented Nt, Eo, Ly, lymphoblasts.

• Increased serum protein.

Calf, 1st Sampling, Chronic Bronchitis, Endoparasites

• Changed RBC size and shape.

• Varying central zone.

• Mid-size Ly.

• Segmented Nt.

• Increased serum protein.

• Platelets.

Calf, 2nd Sampling, Chronic Bronchitis, Endoparasites

• Increased RBC density.

• Changed RBC size and shape.

• Large central zone and unclear margin.

• Small Ly.

• Segmented Nt.

Bull, 6 months old, Purulent Omphalophlebitis, Diarrhea

• Acanthocytes.

• Changes in RBC size.

• Large immature RBC without central pale zone.

• RBC fragments.

• Large Eo.

• Segmented Nt.

• Small Ly.

Goat, 5 years old, Chronic Verminous Bronchopneumonia

• Missing central pale zone.

• Unclear cells.

• Rouleaux formation.

• Segmented and band Nt.

• Eo.

• Platelets.

Sheep, 4 years old, Necrobacillosis, Chronic Verminous Bronchopneumonia, Trichostrongyloidosis

• Large central pale zone.

• Changes in RBC size and shape.

• RBC fragments.

• Rouleaux formation.

• Segmented Nt, Ly.

• Platelets.