Objective: Calculate the percentage composition of different types of WBCs.
Materials Needed: Slides, rack, droppers, blow tube, lancets, spirit, cotton, distilled water with buffer, and stain (Lishman stain).
Goal: Identify and count different types of leukocytes under a microscope.
Types of Stains
Lishman Stain: Used in this experiment.
Components:
Methylene blue: Stains nucleus and basophilic granules
Eosin: Stains cytoplasm and eosinophilic granules
Acetone-free methyl alcohol: Preserves cells and fixes the smear
Gimsa Stain
Wright Stain
Procedure
Blood Collection and Slide Preparation
Sterilize the finger using spirit.
Prick the sterilized ring finger and collect blood on slides (three slides recommended for comparison).
Create a smear using another slide at a 45-degree angle to spread the blood.
Desired smear: Tongue-shaped, covers entire width and at least two-thirds of the slide, uniform thickness, no striations or air patches.
Staining and Fixation
Apply Lishman stain to cover the entire slide and wait for 1-2 minutes.
Dilute stain with water (buffered) to stain the slide; wait 7-8 minutes.
Wash off excess stain gently; allow slides to dry.
Microscopic Examination
Add cedar wood oil to the dried slide and place it under a microscope (oil immersion technique).
Focus the slide by initially raising it until the oil touches the lens, then use fine adjustment.
Count different types of WBCs using a 10x10 grid to record results (L for lymphocytes, N for neutrophils, E for eosinophils, B for basophils, M for monocytes).
Ensure to avoid counting the same cell twice by moving systematically across the slide.
Leukocyte Identification
Neutrophils: Lobed nucleus (3-5 lobes).
Basophils: S-shaped nucleus.
Eosinophils: Goggle-shaped nucleus.
Lymphocytes: Large, round nucleus covering most of the cell.
Monocytes: Kidney or horseshoe-shaped nucleus, larger than RBCs.
Functions of Different WBCs
Neutrophils: Phagocytosis of bacteria & dead cells.
Eosinophils: Detoxify histamines, attack antigen-antibody complexes, numbers increase during allergies.
Basophils: Release histamine and heparin, involved in allergic responses.
Monocytes: Engulf large particles and pathogens; increase during chronic infections.
Additional Information
Arneth Count
Reflects the distribution of neutrophils based on nucleus lobes.
Indicates bone marrow activity: High unlobed neutrophils suggest rapid production (e.g., leukemia), high multi-lobed neutrophils suggest depressed activity.
Differential vs. Absolute WBC Count
Differential WBC Count: Proportion of each type of leukocyte.
Absolute WBC Count: Actual number of each type of WBC, calculated by multiplying differential count percentages with total leukocyte count.
Significance: Helps in identifying infections, anemias, bone marrow activity, and parasitic infections.
Observations from Blood Smears
RBC structure and defects (e.g., anemia).
Presence of parasites (e.g., malaria).
Insight into bone marrow activity via Arneth count.