WBC PP
Unit 2: Hematology
Chapter 10: Evaluating the Blood Film
Instructor: Scott Wilson, DVM
Blood Smears
Purpose: Used to perform the differential white blood cell (WBC) count, estimate platelet numbers, and evaluate morphologic features of WBCs, red blood cells (RBCs), and platelets.
Creation: Recommended to create blood smears even when using an automated hematology analyzer.
Preparing a Blood Film
Materials:
One drop of blood from an EDTA collection tube.
Either a pipette or wooden applicators.
Method:
Blood can be obtained by placing two wooden applicator sticks into the tube, holding them together when withdrawing them.
Preparing a Blood Smear
Place the drop of blood on the frosted end of a clean glass slide.
Write patient information on the frosted end of the slide.
Spreading Method:
Place the end of a second slide against the surface of the first slide at a 30-degree angle and draw it back into the drop of blood.
Once the blood is spread along the width of the spreader slide, push it forward in a steady, even, rapid motion.
Drying:
Immediately air dry the slide.
Maintain the spreader slide at approximately 30 degrees while drawing it back into the blood drop.
Differing Angles
Figure 10-4 shows the difference in slide angle necessary for making blood smears:
A large angle is used for anemic blood (A).
A small angle is used for hemoconcentrated blood (B).
Coverslip Smears
Procedure:
Place one drop of blood in the middle of a clean coverslip.
Place a second coverslip diagonally on top of the first.
Pull coverslips apart in a single smooth motion to create a smear.
Staining Blood Films
Stains Used:
Romanowsky stains, specifically:
Wright stain
Wright-Giemsa stain
Three-step Romanowsky stain (Diff-Quik):
Fixative: 95% methanol
Eosin buffered with an acidic pH – stains basic components such as hemoglobin and eosinophilic granules.
Methylene blue buffered to an alkaline pH – stains acidic components, including leukocyte nuclei.
Staining Procedure:
Rinse between each staining stage.
Avoid dripping water into staining components.
Air-dry after staining.
Best Results:
Fix slide for at least 60 seconds.
Other stages approximately 30 seconds.
No need to dip in and out of stain jars.
Differential Cell Count
Should be performed even if using an automated system because abnormalities can be missed.
Things to Look For:
Nucleated RBCs
Toxic granulation
Platelet clumps
Target cells
Hemoparasites
Systematic Evaluation
Importance of systematic evaluation to avoid counting errors or missing important observations.
Begin with low-power magnification (×100) to assess the general slide.
Scan the entire slide for platelet clumps, large abnormal cells, or microfilariae.
Locate the feathered edge and move to the monolayer, then use high-power magnification (×1000) with oil immersion.
Relative Blood Count
A count of a minimum of 100 WBCs should be recorded and identified.
Record the number of each type of WBC as a percentage using standard mechanical counters to facilitate differential leukocyte counts.
Absolute Values
Importance: Relative percentages may be misleading if the total WBC count is outside of the normal range.
Calculation:
Multiply total WBC count by the percentage of each type.
Example: If there are 80% neutrophils and the total WBC count is 6000/µL, then the absolute value of neutrophils is given by:
White Blood Cells
Categories:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Each cell type plays a role in the body’s defense system, with functions including phagocytosis, antibody production, and modulation of the immune system.
Total concentrations of each type are valuable for diagnosing various diseases.
Neutrophils
Overview:
Most abundant WBC in most mammals.
Nucleus is irregular and elongated; true filaments between lobes are rare.
Normal neutrophils have 3 to 5 nuclear lobes.
Primary Function:
Phagocytosis.
An increase in neutrophil numbers indicates infection or inflammation.
A neutrophil depicted in a blood smear from a normal canine.
Heterophils
Description:
Similar to neutrophils but found in birds, reptiles, and some fish.
A neutrophil from a reptile blood smear is depicted alongside heterophils.
Band Neutrophils
Appearance:
Nucleus is horseshoe-shaped with large, round ends.
If the constriction makes up more than 1/3 of the width of the nucleus, it is classified as a segmented neutrophil.
Example: Depicted canine band neutrophils.
Eosinophils
Characteristics
Nucleus:
Similar to neutrophils but less coarsely clumped chromatin.
Shape and Size Variability:
Varies among species and within species, such as in dogs.
Feline eosinophilic granules are small, elongated, and numerous.
Equine eosinophils contain large, round to oval granules staining orange-red in color.
Function:
Capable of phagocytosis with a primary role in the modulation of the immune system.
Increased eosinophil numbers indicate allergies and parasitic infestations.
Variability in Different Species:
Illustrates differences in eosinophil granule size, shape, and color across species (Canine, Feline, Equine, Bovine).
Basophils
Appearance:
Nuclei similar in appearance to monocytes.
Relatively few in dogs, with granules staining purple to blue-black.
More common in horses and cattle.
In felines, basophils have round granules and stain bright lavender.
Function:
Mediate immune responses.
Increased numbers signify inflammation or infectious conditions.
Normal feline basophil depicted.
Lymphocytes
Characteristics:
Variety of sizes; most abundant WBC in ruminants.
Small lymphocytes in dogs and cats possess slightly indented nuclei with coarsely clumped chromatin and bluish cytoplasm.
Medium to large lymphocytes may have pink-purple granules in the cytoplasm.
Bovine lymphocytes have nucleolar rings.
Major Function:
Production of antibodies; increased lymphocyte counts suggest a viral infection.
Depiction of a small, mature lymphocyte in blood from a normal canine.
Monocytes
Overview:
Largest WBC.
Nuclei may be variably shaped: kidney-bean, elongated, lobed, or amoeboid.
Cytoplasm is blue-gray and may exhibit vacuoles and fine pink granules.
Function:
Primarily phagocytosis; increased numbers can indicate chronic infections.
Depicted are normal canine monocytes alongside neutrophils.
WBC Abnormalities
Pelger-Huet Anomaly
Description:
Characterized by nuclear hyposegmentation; a congenital defect.
Results in hyposegmentation of all granulocyte nuclei.
Depicted: Nuclear hyposegmentation in neutrophils and eosinophils from a dog with Pelger-Huet anomaly.
Nuclear Hypersegmentation
Overview:
A very common change where nuclei have 5 or more lobes.
Can result from aging neutrophils either in vivo or in vitro due to prolonged storage.
Especially common in poodles with macrocytosis.
Example: Canine neutrophil with a hypersegmented nucleus shown.
Toxic Change
Description:
Cytoplasmic basophilia, presence of Döhle bodies, toxic granulation, and gigantism.
These changes are associated with inflammation, infection, or drug toxicity.
Depicted: A toxic neutrophil exhibiting cytoplasmic basophilia and a large Döhle body, while red blood cells are crenated.
Intracytoplasmic Inclusions
Examples include:
Histoplasma capsulatum
Francisella philomiragia
Mycobacterium
Gametocytes of Hepatozoon canis
Amastigotes of Leishmania infantum
Ehrlichia morulae
Depicted: Canine neutrophil containing an Ehrlichia morula.
Atypical Lymphocytes
Description:
These cells have basophilic cytoplasm and cleaved nuclei.
Depicted: An atypical lymphocyte with azurophilic granules in a canine blood smear.
Reactive Lymphocytes (Immunocytes)
Description:
Increased basophilic cytoplasm, more abundant cytoplasm, and sometimes convoluted nuclei.
Usually occurs due to antigenic stimulation.
Depicted: A reactive lymphocyte in a canine blood smear alongside numerous acanthocytes.
Lysosomal Storage Disorders
Overview:
Rare inherited diseases where a substance is abnormally stored in cells.
The stored substance may be seen in leukocytes (typically monocytes, lymphocytes, or neutrophils).
Clinical signs can vary but often affect skeletal or neurologic function.
Lymphocytes may exhibit vacuolation or contain granules; neutrophils may also display granules.
Depicted: Feline lymphocyte containing vacuoles and granules, as well as a neutrophil with toxic granulation.
Birman Cat Neutrophil Granulation Anomaly
Description:
Neutrophils contain fine eosinophilic to magenta granules.
An inherited autosomal-recessive trait where neutrophil function remains normal, and affected cats appear healthy.
Depicted: Cytoplasmic granules associated with Birman cat anomaly.
Chédiak-Higashi Syndrome
Overview:
Neutrophils possess large, fused lysosomes with light pink or eosinophilic cytoplasm.
Approximately 1 in 3 or 4 neutrophils are affected.
Affected animals may bleed due to abnormal platelet function but appear healthy otherwise.
Common among Persian cats, cattle, and foxes.
Siderotic Granules
Description:
Present in neutrophils and monocytes of animals with hemolytic anemia.
Appears similar to Döhle bodies.
Can be differentiated utilizing the Prussian blue stain.
Siderotic granules can occur in RBCs as well (siderocytes).
Smudge Cells
Characteristics
Also known as basket cells; represent degenerative leukocytes that have ruptured.
Depiction: A smudge cell along with several neutrophils seen in a canine blood smear.
Smudge Cells: Additional Information
Karyolysis: Degenerative change to the nucleus due to dissolution of the nuclear membrane, often seen in septic exudates.
Pyknosis: Condensing of the nucleus as the cell dies.
Karyorrhexis: Fragmentation of the nucleus after cell death.
Unit 3: Hemostasis
Chapter 16: Platelet Evaluation
Platelets
Description:
Small cytoplasmic fragments shed from megakaryocytes in bone marrow.
Methods for Evaluation:
Platelet counts
Platelet indices
Platelet function
Conditions:
Thrombocytopenia: Decreased platelet count.
Thrombocytosis: Increased platelet count.
Platelet Counts
Automated Methods:
Automated hematology analyzers may yield inaccuracies due to clumping and overlap.
Manual Counting:
Involves using a chamber or tube with a premeasured volume of diluent and blood, utilizing a hemacytometer for counting.
Observing Morphologic Changes
What to Assess:
Aggregation, giant platelets, reticulated platelets (newly released platelets with high RNA levels).
Depiction: Examples include a giant platelet and a slightly enlarged platelet in a canine patient.
Platelet Estimates
Methodology
Indirect Measurements:
Utilizes the differential blood film, focusing on the monolayer.
Count the number of platelets in at least 10 microscopic fields.
Normal count is 8 to 10 platelets per oil-immersion field.
Multiply the average number by 15,000 or 20,000 to estimate platelet counts.
Alternate Method:
Count the number of platelets per 100 WBCs on the film, then calculate platelet estimates using the formula:
Platelet Indices
Definitions and Measurements
Mean Platelet Volume (MPV):
The mathematical average size of the individual platelets counted by the analyzer.
Increases Indicated:
Increased loss, destruction, or consumption.
Note: Cats typically have larger platelets, making this measure less useful for cats.
Specific breeds like Cavalier King Charles spaniels may display variations.
Exposure to EDTA can also influence this metric.
High MPV typically indicates an adequate bone marrow response, but normal or low does not predict a poor bone marrow response.
Plateletcrit (Thrombocrit):
Measures the percentage of total blood volume comprised of platelets.
Determined by multiplying total platelet count by the mean platelet volume.
Typically less than 1% in most mammals.
Platelet Distribution Width
Purpose:
Assesses variation in platelet size; larger platelets are seen in thrombocytopenia.
Not always correlated with bone marrow response.
Depicted: Histogram showing platelet distribution width.
Other Platelet Tests
Platelet Function Tests:
Evaluate platelet functionality.
Thrombopathia:
Refers to alterations in platelet function.
Unit 3: Hemostasis
Chapter 14: Principles of Blood Coagulation
Hemostasis
Definition:
The body’s ability to maintain the integrity of blood and blood vessels.
Components Involved:
A complex interplay of pathways, platelets, and coagulation factors.
Mechanical Phase of Hemostasis
Initiation:
Triggered when a blood vessel ruptures or tears.
Exposed subendothelium of vessel introduces a charged surface.
Platelet Action:
Platelets adhere to this charged surface, undergo morphological and physiological changes, adhere to one another, and the endothelium.
Phosphatidylserine (PS) Exposure:
Activated platelets expose PS on their outer membrane, facilitating further coagulation processes.
von Willebrand Factor Requirement:
Stabilizes the platelet plug formed during adhesion and aggregation.
Triggering the Chemical Phase:
The actions of the mechanical phase set off the chemical phase.
Chemical Phase of Hemostasis
Coagulation Cascade:
Involves various factors and consists of intrinsic and extrinsic pathways, leading to the formation of a fibrin mesh or clot.
Coagulation Factors:
Table 14-1 summarizes important factors and their synonyms:
Factor I: Fibrinogen
Factor II: Prothrombin
Factor III: Tissue factor
Factor IV: Calcium
Factor V: Proaccelerin
Factor VII: Proconvertin
Factor VIII: Antihemophilic factor
Factor IX: Christmas factor, plasma thromboplastin
Factor X: Stuart factor
Factor XI: Plasma thromboplastin antecedent
Factor XII: Hageman factor
Factor XIII: Fibrin-stabilizing factor, prekallikrein
Initial Reactions for the Chemical Phase
Initiating factors including tissue factor (TF) binding to FVIII (plasma) lead to activation of FX.
The cascade amplifies thrombin generation, resulting in increased clot formation.
Factors involved:
FX
Intrinsically activated factors including XI, VIII, and XIII
Calcium ions essential for the reactions
Functions of Thrombin During Later Stages of Chemical Hemostasis
Actions:
Recruitment and activation of additional platelets.
Converts soluble fibrin into an insoluble (cross-linked) form.
Breakdown of Fibrin:
Plasminogen is converted into plasmin, leading to fibrin degradation products (FDPs) including d-dimers and cross-linked FDPs.
Summary of the Chemical Phase of Hemostasis
Coagulation Cascade Phases:
Intrinsic pathway characterized by factors such as FXII, FXI, FIX, and FVIII.
Extrinsic pathway initiated by tissue factor.
Common pathway leading to the formation of fibrinogen converted to fibrin.
Fibrinolysis resulting in the degradation of fibrin and production of FDPs including d-dimers.
Unit 3: Hemostasis
Chapter 17: Coagulation Testing
Coagulation Tests
Purpose:
Evaluate specific phases of the coagulation process and utilize specialized instruments.
Preferred over manual tests due to variability in results.
Note:
Test results may be altered if platelet counts are low.
Coagulation Tests: Intrinsic and Extrinsic Systems
Diagram: Traditional representation highlighting intrinsic, extrinsic, and common coagulation pathways detailing their associated factors.
Buccal Mucosa Bleeding Time
Purpose:
Primary assay for detecting abnormalities in platelet function.
Required Supplies:
Spring-loaded lancet, blotting paper or filter paper, stopwatch, tourniquet.
Procedure:
Sedate or anesthetize the patient in lateral recumbency.
Tie the upper lip back and make a 1-mm-deep incision.
Blot the incision site every 5 seconds until bleeding stops.
Normal Range:
1 to 5 minutes; prolonged bleeding suggests platelet dysfunction or deficiencies in von Willebrand factor or thrombocytopenia.
Activated Clotting Time (ACT)
Purpose:
Can evaluate every clinically significant clotting factor except for Factor VII.
Method:
Utilize a vacutainer containing diatomaceous earth or kaolin, which triggers coagulation pathways.
Prewarm the tube to 37° C.
Collect 2 mL via venipuncture and time from collection to presence of a clot.
Normal Range:
60 to 90 seconds; automated analyzers may be available.
Whole Blood Clotting Time
Lee-White Method:
An older test not commonly performed due to the sensitivity of activated clotting tests.
3 mL of blood is drawn into a plastic syringe and timed from collection.
Blood is placed immediately into three pre-prepped tubes and incubated at 37° C.
Measure clotting time from collection to clot formation in the third tube.
Normal Range:
Dogs: 1 to 10 minutes; Horses: 4 to 15 minutes; Cattle: 10 to 15 minutes.
Activated Partial Thromboplastin Time (aPTT)
Purpose:
Evaluates intrinsic and common clotting mechanisms.
Used Equipment:
Coag Dx™ analyzer or handheld analyzers.
Factors Affecting Results:
Various clotting disorders and addition of heparin.
Prothrombin Time Test
Definition:
One-stage prothrombin time (OSPT), typically performed with automated analyzers.
Purpose:
Evaluates intrinsic and common coagulation pathways.
Normal Range:
Dogs: 7 to 10 seconds; prolonged times indicate potential severe liver disease, disseminated intravascular coagulation (DIC), or hereditary/acquired deficiencies.
Vitamin K Deficiency:
Significant factor causing prolonged prothrombin time.
Clot Retraction Test
Objective:
Evaluates platelet numbers and function as well as intrinsic and extrinsic pathways.
Methodology:
Draw blood into a plain sterile tube and incubate at 37° C.
Initial Assessment:
Examine at 60 minutes and periodically over 24 hours.
Clots should be evident in 60 minutes and retract within approximately 4 hours.
Marked retraction should be observed at 24 hours.
Note: Does not provide information about the underlying source of coagulopathy.
Fibrinogen Determination
Methods:
Automated methods are not commonly performed in hospitals.
Manual method:
Two hematocrit tubes are centrifuged for PCV.
Determine total solids in one tube; incubate the other at 58° C for 3 minutes before re-centrifugation.
Measure total solids again; calculate:
Fibrinogen mg/dL formula:
PIVKA
Definition:
Proteins induced by vitamin K absence; necessary for activating Factors II, VII, IX, and X (2, 7, 9, 10).
Mechanism:
Upon vitamin K deficiency, precursor proteins build up and can be detected by PIVKA.
More sensitive than prothrombin levels.
Timelines:
May be prolonged within 6 hours of rodenticide ingestion; 24 hours for prothrombin; and 48 hours for aPTT.
D-Dimer and Fibrin Degradation Products
Purpose:
Evaluate tertiary hemostasis (fibrinolysis).
Formed as a clot degrades, useful in identifying conditions such as DIC, liver failure, trauma, and hemangiosarcoma.
Testing:
In-house testing for D-dimer analysis is often done through immunoassays, which are more specific than fibrin degradation products (FDPs).
von Willebrand Factor
Role:
Required for platelet adhesion.
Testing:
Typically performed when platelet function defects are suspected.
Immunoassays are conducted primarily in reference laboratories.
Coagulation Factor Assays
Purpose:
Conducted in reference laboratories to ascertain disorders due to hereditary** or acquired conditions that may alter clotting factors.
Unit 3: Hemostasis
Chapter 18: Disorders of Hemostasis
Hemostatic Defects
Causes of Bleeding Disorders:
Congenital defects
Acquired defects
Most disorders are secondary to another disease process.
Types:
Primary coagulation disorders resulting from congenital defects.
Clinical Signs
Signs associated with both congenital and acquired defects:
Superficial petechiae
Ecchymotic hemorrhage
Bilateral epistaxis
Melena
Prolonged bleeding at injection sites or incisions.
Function Deficiencies or Deficiencies:
Typically present before the animal reaches 6 months of age.
Example: Petechiae may signify a coagulation abnormality, depicted with epistaxis in a Saint Bernard.
Hereditary Coagulation Factor Disorders
Includes various factor deficiencies with Hemophilia A (Factor VIII deficiency) being the most common inherited condition in dogs.
Hemophilia D (Christmas disease) refers to Factor IX deficiency.
Table 18-1 summarizes common inherited coagulation disorders in various breeds, highlighting their affected breeds:
Prothrombin deficiency: Cocker Spaniel, Beagle
Factor VII deficiency: Beagle, Malamute
Factor VIII deficiency: Various breeds (Hemophilia A)
Factor IX deficiency: Various breeds (Hemophilia B)
Factor X deficiency: Cocker Spaniel
Factor XI deficiency: Great Pyrenees, English Springer Spaniel
Factor XII deficiency: Poodle, Shar Pei
von Willebrand Disease
Overview
Description:
The most common inherited coagulation disorder in domestic animals, marked by a decrease in von Willebrand factor, a large glycoprotein aiding in platelet aggregation during the coagulation process.
Affected Animals:
More prevalent in Doberman pinschers and other canines, rabbits, and swine.
Types
Type 1:
Characterized by low levels of normal structure (autosomal dominant with incomplete penetrance).
Type 2:
Low levels of vWF with abnormal structure (dominant inheritance).
Type 3:
Near absence of vWF (autosomal recessive); Types 2 and 3 present the most severe bleeding cases.
Acquired Coagulation Disorders
Types
Thrombocytopenia:
The most common coagulation disorder seen in veterinary practices due to decreased platelet numbers.
Causes include viral, bacterial, parasitic infections, and certain medications like Aspirin and Acetaminophen.
Vitamin K Deficiency:
Required for the synthesis and activation of Factors II, VII, IX, and X.
Can be due to dietary reasons or bile duct obstruction.
Ingestion of rodenticides or moldy sweet clover may induce this deficiency.
Common signs include lethargy, anorexia, dyspnea, ecchymosis, petechiae, and hemarthrosis.
Diagnostic tests include PT, aPTT, ACT, and PIVKA.
Recovery may require several weeks of treatment.
Disseminated Intravascular Coagulation (DIC):
Associated with many devastating conditions like trauma, septicemia, various viral and parasite infections, pancreatitis, and toxin exposure.
Symptoms include systemic hemorrhage or microvascular thrombosis.
Table 18-4 illustrates expected laboratory test results for common bleeding disorders, highlighting specific alterations in BMBT, ACT, PT, aPTT, platelets, fibrinogen, FDPs, and D-Dimers for different conditions.