3. Blood - ALL

Blood - Introduction

  • Functions of Blood: Two major functions:

    • Transport of oxygen

    • Protection against microorganisms

  • Objectives:

    • Examine blood constituents

    • Study leucocyte immunological functions

    • Study antigen-antibody interactions

  • Lab Safety:

    • Wear gloves/lab coats when handling blood or Drabkin's solution

    • Handle only your own blood samples

    • Dispose of contaminated materials in yellow Bio-hazard bucket

    • Report spills; clean with Bleach and Accel solutions

    • Notify lab instructor if feeling light-headed/nauseated

    • Wash hands before leaving; no food or drink allowed

Blood Cell Types Demonstration

  • Microscope Examination: Identify human blood stained with Wright's stain:

    1. Erythrocyte (red blood cell)

    2. Agranular leukocytes:

      • a) Monocyte

      • b) Lymphocyte

    3. Granular leukocytes:

      • a) Basophil

      • b) Heterophil or Neutrophil

      • c) Acidophil or Eosinophil

    4. Platelets

  • Comparison Questions:

    • Compare sizes of erythrocytes and leukocytes

    • Sites of formation of erythrocytes and leukocytes

    • Tissues/organs involved in erythrocyte production during embryonic/fetal life

    • Define leukopenia

Blood - Hemoglobin

  • Experimental Procedures: Determination of Hemoglobin Concentration

    1. Add 5.0 mL Drabkin's solution to a test tube (Caution: Contains cyanide)

    2. Gather materials: heparinized capillary tubes, alcohol swab, microfuge tube, and Microtainer lancet

    3. Blood Sample Collection:

      • Partner holds donor's finger; swab with alcohol

      • Puncture skin with lancet and fill capillary tubes by capillary action

      • Transfer blood to microfuge tube

    4. Mixing:

      • Cap and invert tube to mix; dispense 20 μL of blood into Drabkin's solution

      • Ensure thorough mixing to prevent clotting

    5. Measurement:

      • Let sit for 10 min before measuring absorbance using Spectronic 200

      • Record absorbance for standard (16 g/dL)

      • Analyze own sample absorbance

    6. Calculating Hemoglobin Concentration:

      • Formula: Hemoglobin concentration (g/dL) = A(sample)/A(standard) x 16

    7. Normal Ranges:

      • Males: 16 ± 2 g/dL

      • Females: 14 ± 2 g/dL

      • Average for humans: 140 - 180 g/L (males), 120-160 g/L (females)

      • Average for sheep: 90-150 g/L

    8. Dispose of waste properly.

Blood - Red Cell Count

  • Experimental Procedures: Red Blood Cell Count

  • Counting Chamber: Features of the disposable slide:

    • H-shaped trough with central platforms

    • Blood layer thickness of 0.1 mm

    • Rulings divided for counting areas (Total area = 400 total squares)

  • Red Cell Preparation:

    • Make 200X dilution of blood; draw up 20 μL, combine with 4 mL diluting fluid

  • Filling the Counting Chamber:

    1. Draw up 20 μL of diluted blood, release under coverslip

    2. Allow cells to settle 2 min before counting

  • Counting Method:

    1. Examine at 10X, then 40X objective

    2. Count cells in 80 small squares total (using counters)

  • Calculating Red Blood Cells per Cubic mm:

    • Formula: Total cells counted x 5 (area) x 10 (volume) x 201 (dilution)

    • Estimates for sheep and human blood cell counts

Blood - Hematocrit

  • Experimental Procedures: Finding Hematocrit

    1. Collect blood into a heparinized capillary tube

    2. Centrifuge tubes for 5 min at 10,000 rpm

    3. Measure heights of plasma and red cells

    4. Calculate Hematocrit:

      • Formula: Hct(L/L) = Height of red cells (mm) / (Height of red cells + plasma (mm))

    5. Normal Values:

      • Males: 0.40 - 0.54

      • Females: 0.37 - 0.47

      • Sheep: 0.27 - 0.45

Blood - MCV and MCH Calculation

  • Experimental Procedures: Calculating MCV and MCH

  • Mean Corpuscular Volume (MCV):

    • Formula: MCV (femtolitres) = Hematocrit x 1000 / RBC count (10^6/L)

    • Normal ranges for humans: 87±5 fL; for sheep: 28-40 fL

  • Mean Corpuscular Hemoglobin (MCH):

    • Formula: MCH (picograms) = Hemoglobin (g/L) / RBC count (10^6/L)

    • Normal ranges for humans: 29±2 pg; for sheep: 31-34 pg

Blood - Hemostasis Background

  • Hemostasis Overview:

    • Blood clots through fibrinogen converting to insoluble fibrin via thrombin

    • Fibrin strands trap blood cells forming clots

    • Prothrombin converts to thrombin guided by clotting factors and calcium ions

  • Clotting Pathways:

    • Intrinsic Pathway: Triggered by collagen exposure; involves several reactions

    • Extrinsic Pathway: Initiated by tissue thromboplastin, shortcuts intrinsic pathway

Blood - Hemostasis Procedures

  • Experimental Procedures:

    1. Intrinsic Pathway: Mix excess calcium with sheep blood and record clot time

    2. Extrinsic Pathway: Mix tissue thromboplastin and calcium with sheep blood and record clot time

  • Bleeding vs Coagulation Time:

    • Bleeding time measures all hemostasis forms

    • Coagulation time focuses on intrinsic pathway

Blood - Immunity Background

  • Non-specific Immunity:

    • Neutrophils and monocytes defend against infections via diapedesis and chemotaxis

    • Phagocytosis as a primary defense mechanism

  • Specific (Acquired) Immunity:

    • Lymphocytes (B-cells and T-cells) identify and attack specific invaders

    • Antibodies produced by B-cells aid in defense

Blood - Immunity Procedures (Leukocyte Count)

  • White Cell Preparation:

    • Use 2% acetic acid to lyse red cells

    • Create a 20X dilution; mix and fill counting chamber

  • Counting Procedure:

    1. Count nuclei using 40X, maintaining slide integrity

    2. Calculate leukocyte/mm using given formula

Blood - Immunity Procedures (Leukocyte Identification)

  • Identification of Granular and Agranular Leukocytes:

    1. Prepare blood smear and air dry

    2. Stain with Giemsa and Wright’s stain

    3. Observe under microscope to identify leukocyte types

  • Normal Percentages:

    • Neutrophils: 62%, Eosinophils: 2%, Basophils: 1%, Monocytes: 5%, Lymphocytes: 30%

Blood - Immunity Procedures (Blood Typing)

  • ABO Typing:

    • Presence of A and B antigens correlates with blood groups

    • Perform agglutination test with anti-A and anti-B sera

  • Rh Factor Typing:

    • Determine presence (Rh-positive) or absence (Rh-negative) of D antigen

Blood - Clean Up

  • Cleanup Procedures:

    1. Clean microscope parts

    2. Turn off equipment

    3. Dispose of all used materials in designated waste buckets.