Unit 2: Blood Cells

I. Orientation

Blood-cell morphology is the study of the size, shape, colour, inclusions, and nuclear characteristics of formed elements in a stained peripheral blood smear. These features are examined microscopically to distinguish erythrocytes, leukocytes, and platelets and to recognize physiological variation or disease-associated abnormalities.

• Specimen: Fresh venous blood collected in an EDTA tube is commonly used because EDTA prevents clotting while preserving cellular detail.
• Peripheral smear: A thin film of blood is spread on a clean glass slide, air-dried, fixed, and stained before microscopic examination.
• Romanowsky staining: Wright, Wright–Giemsa, or Leishman stains contain acidic and basic dyes that produce contrasting colours in cytoplasm, granules, nuclei, and haemoglobin.
• Microscopic sequence: Examine the smear first under low power to assess thickness and distribution, then under high power or oil immersion for cellular identification.
• Normal distribution: Erythrocytes are the most numerous cells; leukocytes are fewer; platelets are small cytoplasmic fragments scattered between red cells.
• Morphological criteria: Identification depends on diameter, outline, cytoplasmic staining, nuclear shape, chromatin pattern, granules, and intracellular inclusions.
• Reference conditions: Morphology is best assessed in the monolayer, where red cells are close together but not overlapping excessively.
• Interpretive principle: A finding is meaningful only when correlated with the smear quality, blood-cell count, patient information, and possible preparation artifacts.

II. Morphology of Blood Cells — Identification in a stained peripheral smear

Blood-cell morphology provides a visual description of the formed elements of blood. The laboratory worker compares observed cells with normal structural features before recording abnormalities.

A. Purpose and microscopic principle

The purpose of the examination is to identify normal blood cells and detect changes that may indicate altered production, maturation, destruction, or immune response.

• Cellular components: The smear contains erythrocytes, leukocytes, and platelets. Plasma is largely removed or spread into a thin background during preparation.
• Staining principle: Basic dyes such as methylene blue stain acidic structures, including nuclear chromatin, blue-purple; acidic eosin stains basic proteins, including haemoglobin, pink to orange.
• Magnification: A 10× objective evaluates smear quality, a 40× objective surveys cell distribution, and a 100× oil-immersion objective gives approximately 1000× total magnification with a 10× eyepiece.
• Monolayer location: The optimal area is just behind the feathered edge, where erythrocytes form a single layer and central pallor is visible.
• Counting context: A differential leukocyte count classifies a defined number of leukocytes, commonly 100 or 200, as neutrophils, lymphocytes, monocytes, eosinophils, or basophils.
• Morphological report: Findings should describe the red-cell population, leukocyte abnormalities, platelet estimate, and significant inclusions rather than relying only on vague terms such as “abnormal cells.”

B. Study of morphology of blood cells

The study of morphology of blood cells involves recognizing each formed element by its characteristic size, shape, staining pattern, and internal structures.

• Erythrocytes: Normal red blood cells are anucleate, flexible biconcave discs measuring approximately 7–8 µm in diameter.

  • Colour: They appear pink to salmon because of haemoglobin, with a central area of pallor occupying about one-third of the cell diameter.
  • Shape: The regular round outline and central pallor reflect the biconcave structure; cells lacking central pallor may be spherocytes.
  • Size variation: Cells smaller than normal are microcytes, while cells larger than normal are macrocytes. A mixed population shows anisocytosis.
  • Shape variation: Poikilocytosis means variation in red-cell shape. Examples include ovalocytes, target cells, sickle-shaped cells, teardrop cells, and fragmented cells.
  • Inclusions: Howell–Jolly bodies are small, round, dark-purple nuclear remnants; basophilic stippling consists of fine blue granules representing aggregated ribosomal material.
  • Immature forms: Reticulocytes may show a faint bluish tinge, called polychromasia, because residual RNA stains blue-gray.

• Neutrophils: Neutrophils usually measure about 10–15 µm and commonly constitute approximately 50–70% of circulating leukocytes in adults.

  • Nucleus: The nucleus has 2–5 connected lobes with coarse, dark-purple chromatin.
  • Cytoplasm: Cytoplasm is pale pink or lilac and contains numerous fine, inconspicuous specific granules.
  • Maturity: Increased band forms show a curved or horseshoe-shaped nucleus without distinct lobulation; toxic granulation appears as coarse dark granules in reactive states.
  • Function link: Their multilobed nucleus and granule-rich cytoplasm support rapid movement and phagocytosis during acute inflammation.

• Lymphocytes: Small lymphocytes are approximately 7–10 µm, whereas large lymphocytes may reach 12–15 µm; they commonly represent about 20–40% of leukocytes.

  • Nucleus: The nucleus is round or slightly indented, occupies most of the cell, and contains dense, clumped chromatin.
  • Cytoplasm: A small rim of clear to pale-blue cytoplasm surrounds the nucleus. Occasional azurophilic granules may be present.
  • Reactive forms: Reactive lymphocytes are larger, with abundant deep-blue cytoplasm that may mold around adjacent red cells; this appearance is associated with antigenic stimulation.

• Monocytes: Monocytes are the largest normal circulating leukocytes, commonly measuring about 15–20 µm and comprising approximately 2–8% of leukocytes.

  • Nucleus: The nucleus is often kidney-shaped, folded, or horseshoe-shaped, with relatively fine chromatin.
  • Cytoplasm: Abundant gray-blue cytoplasm may contain fine reddish or lilac granules and occasional vacuoles.
  • Recognition clue: Their large size, folded nucleus, and gray cytoplasm distinguish them from the more compact, dark lymphocyte.

• Eosinophils: Eosinophils usually measure 12–17 µm and normally account for about 1–4% of leukocytes.

  • Nucleus: The nucleus is typically bilobed, with lobes connected by a thin filament.
  • Granules: Large, uniform, orange-red granules fill the cytoplasm and may partly obscure the nucleus.
  • Functional association: Their morphology corresponds to participation in allergic reactions and responses to helminthic parasites.

• Basophils: Basophils are approximately 10–14 µm and are usually less than 1% of circulating leukocytes.

  • Nucleus: The nucleus is often bilobed or irregular but may be difficult to see.
  • Granules: Large, coarse, dark-blue to purple granules frequently overlay the nucleus.
  • Recognition clue: The combination of very dark granules and low frequency makes basophils easy to overlook or confuse with stain precipitate.

• Platelets: Platelets are anucleate cytoplasmic fragments derived from megakaryocytes and typically measure 2–4 µm.

  • Appearance: A normal platelet has a pale-blue peripheral hyalomere and a central purple granular zone containing azurophilic granules.
  • Distribution: They occur singly or in small groups between erythrocytes; approximately 7–20 platelets may be seen per oil-immersion field in a well-prepared smear, although field estimates vary.
  • Abnormal size: Large or giant platelets may approach the diameter of a red cell and can indicate increased platelet turnover or altered megakaryocyte production.
  • Artifacts: Platelet clumps, especially near the feathered edge, may cause a falsely low automated platelet count.

C. Smear preparation and staining factors

Correct preparation is essential because poor technique can alter apparent blood-cell morphology.

• Drop size: A small, well-mixed blood drop produces a suitable smear; an excessively large drop creates a thick film with overlapping cells.
• Spreader angle: An angle near 30–45° usually produces an even film. A lower angle creates a longer, thinner smear, whereas a higher angle produces a shorter, thicker smear.
• Drying: Rapid air-drying preserves cell outlines. Slow drying may produce crenation, vacuolation, or distorted leukocyte morphology.
• Fixation: Methanol fixation preserves cellular structure and prevents cells from washing away during staining.
• Stain balance: Excessively acidic staining makes cells too red, while excessive alkalinity makes them too blue. The exact staining time and dilution depend on the selected stain.
• Smear edge: The feathered edge may concentrate large cells, platelet clumps, and abnormal cells, so it should not be used alone for differential assessment.
• Common artifacts: Stain precipitate appears as irregular dark particles; rouleaux appears as stacked red cells; crenated erythrocytes have evenly spaced projections caused by drying or osmotic effects.

D. Applications and limitations

Morphological examination supports diagnosis and quality control, but it must be interpreted with quantitative laboratory results and clinical findings.

• Anaemia assessment: Microcytosis, hypochromia, macrocytosis, polychromasia, or abnormal shapes can suggest different red-cell production or destruction patterns.
• Infection and inflammation: Neutrophilia, band forms, toxic granulation, reactive lymphocytes, or monocytosis may accompany inflammatory or infectious processes.
• Haematological disease: Blasts, marked anisopoikilocytosis, abnormal lymphoid cells, or severe platelet abnormalities require further laboratory investigation.
• Malaria and parasites: Thin smears may demonstrate intraerythrocytic organisms, while appropriate thick-film methods increase detection sensitivity.
• Quality limitation: Cell distortion from delayed processing, incorrect anticoagulant-to-blood ratio, poor staining, or an unsuitable smear area can imitate disease.
• Counting limitation: A 100-cell differential has greater sampling variation than a 200-cell differential, particularly when rare cells such as basophils or blasts are being assessed.
• Confirmation requirement: A suspected abnormal cell should be reviewed in another smear region and, where appropriate, confirmed by repeat testing, special stains, flow cytometry, or other diagnostic methods.
• Safe practice: Blood is potentially infectious; gloves, laboratory coats, sharps precautions, correct disposal, and decontamination are required during smear preparation and microscopy.