Principles and Practice of Clinical Hematology: Microscopic Examination of Peripheral Blood

Hematology

Definition and Scope

Hematology is the branch of biology and medicine concerned with the study of blood, blood-forming tissues, and the formed elements of blood. This includes red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs).

• Formed Elements: The cellular components of blood, including erythrocytes (RBCs), leukocytes (WBCs), and thrombocytes (PLTs).

• Quantitative Analysis: Refers to counting the number of each type of cell present in the blood.

• Qualitative Analysis: Involves assessing the appearance and morphology of each cell type.

Additional info: Hematology is essential for diagnosing and monitoring diseases such as anemia, infections, and blood cancers.

Microscopic Examination of the Peripheral Blood Film

Purpose and Overview

Microscopic examination of peripheral blood is a fundamental technique in clinical hematology. It involves preparing, staining, and examining a thin smear of blood on a glass slide to evaluate the morphology and quantity of blood cells.

• Preparation: A drop of blood is spread thinly across a slide to create a 'blood film' or 'smear.'

• Staining: Special stains are applied to differentiate and visualize RBCs, WBCs, and PLTs.

• Evaluation: The morphology (shape, size, color) of blood cells is assessed, and the percentage of each WBC type is calculated (WBC differential).

Sources of Blood for Blood Film

• Capillary Blood: Obtained from a finger or heel puncture, suitable for morphological examination of white and red cells.

• Venous Blood: Most hematology work uses venous blood, often collected in tubes containing EDTA (an anticoagulant).

• Sample Handling: Only the drop of blood should touch the slide. If using EDTA blood, the smear should be prepared within 2 hours to prevent artifacts.

Additional info: Proper sample collection and handling are critical to avoid cell distortion or artifacts that can affect diagnosis.

Preparation of the Blood Film

The process of making a blood smear involves spreading a drop of blood across a slide using another slide as a spreader. The goal is to create a film that transitions from thick to thin, ending in a 'feathered edge' where cells are well separated for examination.

• Key Steps:

  1. Place a small drop of blood near one end of a clean slide.

  2. Hold a spreader slide at a 30-45° angle and back it into the drop.

  3. Allow the blood to spread along the edge, then push the spreader smoothly forward.

  4. Allow the smear to air dry.

• Quality Features: A good smear has a gradual transition from thick to thin, a feathered edge, and no streaks or clumps.

Staining the Blood Smear

Staining is essential for differentiating cell types and visualizing cellular details. The most common stains are Romanowsky-type stains, such as Wright's stain and Diff-Quik.

• Wright's Stain:

  • Methanol: Fixes and preserves cell structures.

  • Methylene Blue: A basic dye that stains acidic cell components (e.g., nuclei) blue.

  • Eosin: An acidic dye that stains basic cell components (e.g., hemoglobin, cytoplasm) red-orange.

• Diff-Quik Staining Procedure:

  1. Make smears and allow to dry.

  2. Dip slide in fixative five times (one second each).

  3. Dip in Stain 1 (eosin) five times (one second each).

  4. Dip in Stain 2 (methylene blue) five times (one second each).

  5. Rinse slide in water and allow to dry.

Additional info: Proper staining is crucial for accurate identification of cell types and detection of abnormalities.

Microscopic Examination Techniques

Examination of the stained blood film is performed using a light microscope with different objectives for various purposes.

• Low-Power Objective (10x):

  • Assess overall smear quality and staining.

  • Estimate RBC and WBC counts.

  • Scan for abnormal cells or platelet clumps.

• Oil-Immersion Objective (100x):

  • Detailed examination of erythrocyte and leukocyte morphology.

  • Estimation of platelet count and evaluation of morphologic changes.

  • Differential count of leukocytes (identifying and counting at least 100 WBCs).

Storage of Blood Smears

• Store slides in a dark, dust-free box to protect from scratches.

• A permanent coverslip can be applied for long-term storage, though this is rarely necessary in clinical labs.

Erythrocyte (Red Blood Cell) Alterations

Characteristics to Note

When examining erythrocytes, several features are assessed to detect abnormalities that may indicate disease.

• Color (Staining Reaction): Indicates hemoglobin content.

  • Normochromic: Normal color and central pallor.

  • Hypochromic: Increased central pallor, less hemoglobin (seen in iron deficiency anemia).

• Size (Anisocytosis): Variation in RBC size.

  • Macrocytosis: RBCs larger than normal.

  • Microcytosis: RBCs smaller than normal.

• Shape (Poikilocytosis): Variation in RBC shape.

• Structure and Inclusions: Presence of abnormal structures or inclusions within RBCs.

• Artifacts and Distribution: Abnormal patterns or artifacts due to preparation errors.

Clinical Significance of Erythrocyte Alterations

Alterations in erythrocyte morphology are associated with various diseases, especially anemia. Anemia is a condition characterized by decreased oxygen-carrying capacity of the blood, leading to reduced oxygenation of tissues and organs.

• Anemia: Not a single disease, but a condition with many causes. Proper diagnosis of the type and cause is essential for effective treatment.

Leukocytes (White Blood Cells)

Differential Count and Morphology

The differential count involves identifying and counting at least 100 WBCs to determine the percentage of each type. Morphologic examination helps detect abnormal or immature cells.

• Mature WBCs: All WBCs in circulating blood should be mature under normal conditions.

• Leukocyte Alterations:

  • Leukocytosis: WBC count above normal (may indicate infection, inflammation, or leukemia).

  • Leukopenia: WBC count below normal (may indicate bone marrow failure, severe infection, or autoimmune disease).

Summary Table: Key Blood Cell Alterations

Parameter	Normal	Alteration	Clinical Significance
RBC Color	Normochromic	Hypochromic	Iron deficiency anemia
RBC Size	Normocytic	Microcytic/Macrocytic	Thalassemia, megaloblastic anemia
RBC Shape	Disc-shaped	Poikilocytosis	Sickle cell anemia, hereditary spherocytosis
WBC Count	Normal range	Leukocytosis/Leukopenia	Infection, leukemia, bone marrow disorders