Leukopoiesis and Leukocytes

• Divided into myelopoiesis and lymphopoiesis, leukopoiesis refers to the production of white blood cells.

Leukocytes in Normal Peripheral Blood Smear (PBS)

• Granulocytes consist of neutrophils, basophils, and eosinophils.
• Agranulocytes consist of monocytes and lymphocytes.
• Neutrophil bands (1–2%) may be normally seen in PBS; >5% signifies ongoing infection.

Neutrophils

• Characterized by 2-5 lobes (average 3) connected by a narrow filament
• Nuclear indentation is greater than half the diameter of the nucleus
• N/C ratio is 1:3 - 1:5
• Exhibit a dark purple color
• Heavily clumped chromatin pattern with no nucleoli when mature
• Light pink to bluish cytoplasm with evenly distributed pink to rose violet granules
• Primary (azurophilic) granules is about 1/3 of the granules
• Potent hydrophilic enzymes are elastase and myeloperoxidase (MPO)
• MPO is the most important primary granule content
• Secondary (specific) granules account for approximately 2/3 of the granules
• Iron-binding lactoferrin is included in secondary granules
• Gelatinase is included in tertiary granules

Eosinophils

• Possess 2-3 lobes (rarely 3)
• N/C ratio is 1:3 - 1:5
• Exhibit dark purple color with heavily clumped chromatin pattern and lack nucleoli when mature
• Exhibit pink to blue cytoplasm
• Displays colorless Henry’s granules
• Display many large, round, uniform, reddish-orange granules with a strong affinity for acid stains (eosin)

Basophils

• Possess 2 lobes that are usually obscured by granules with N/C ratio of 1:3 - 1:5
• Exhibit dark purple color but paler than the granules
• Heavily clumped chromatin pattern when mature and lack nucleoli
• Display light pink to blue cytoplasm
• Few dark blue-black granules, containing Henry's mauve
• Exhibit large granules with a strong affinity for basic stains; water-soluble

Monocytes

• Macrophages (tissue) are larger, with oval, egg-shaped nucleus with reticular or dispersed chromatin, and visible nucleoli
• Perinuclear (Golgi) zone might be present
• Horseshoe-shaped, deeply indented, or partially lobulated; brain-like convolutions due to nuclear folding
• N/C ratio of 1:1 with a dark purple color
• Stains less densely than other leukocytes
• Exhibit fine delicate strands of chromatin, arranged in linear form, with light spaces between strands
• Mature monocytes lack nucleoli
• Blue-gray, finely granular cytoplasm with ground glass appearance, abundant and irregular at cell margins
• May contain occasional vacuoles and blunt pseudopods with ingested red cells, debris, pigments, or bacteria
• Indicative of active infection
• Display many fine, dust-like bluish, red to purple azurophilic granules

Lymphocytes

• Exhibit round or slightly indented, eccentric nucleus
• N/C ratio of 3:1 with deep purple-blue color
• Coarse and clumped chromatin (dark blue with Wright’s stain; parachromatin: lighter stained streaks
• Sky blue to deep blue cytoplasm (Rodak’s: Robin’s egg blue)
• Clear perinuclear (Golgi) zone and scant, usually non-granular
• Display few azurophilic granules

Granulopoiesis

• Granulocytes have granules in their cytoplasm with segmented or lobulated nuclei.
• GMP (common progenitor cell for neutrophils and monocytes) divides into progenitors for granulocytes (CFU-G) and monocytes (CFU-M).
• CFU-G and CFU-M cells produce myeloblasts and monoblasts respectively when stimulated by colony-stimulating factors.

Myeloblast

• Round-shaped with reddish-purple color and delicate nuclear membrane
• N/C ratio: 7:1 – 4:1
• Chromatin is fine, delicate, and dispersed
• 2-5 distinct and pale blue nucleoli
• Pale to Deep Purple cytoplasm with a lighter staining adjacent to nucleus
• Varying amounts of granules depending on the type
• Type 1: NO visible granules under a light microscope
• Type 2: Number of granules does NOT exceed 20 per cell where scattered azurophilic granules in the cytoplasm
• Type 3: More than 20 granules that do not obscure the nucleus

Promyelocyte

• Round or oval with central or eccentric
• Purple color, may mimic the appearance of a plasma cell
• N/C ratio: 5:1 - 2:1
• Relatively fine chromatin, becoming coarser
• Paranuclear halo or “hof” is usually seen, except in malignant promyelocytes of acute promyelocytic leukemia
• 2-3 nucleoli (varying from visible to indistinct), may be obscured by granules
• Evenly basophilic (bluish) cytoplasm
• Lighter staining adjacent to the nucleus where there are few to many dark blue or reddish-purple primary granules

Myelocyte

• Round, oval or flattened on one side (D-shaped) with dark purple color
• N:C ratio: 3:2 - 3:1 and coarser Chromatin pattern
• Early myelocyte may have visible nucleoli
• Pinkish blue cytoplasm with variable numbers of primary/azurophilic/non-specific granules
• Secondary/ specific granules are small and pinkish to reddish
• In early myelocytes, there are many azurophilic granules with few specific granules, where as Late myelocyte have few azurophilic granules where there are many specific granules

Metamyelocyte

• Indented, kidney-shaped or peanut-shaped
• Indentation should not reach more than half of the nucleus
• Exhibit Dark purple color
• N/C ratio: 7:3 - 1:1 and coarse, condensed, blue-black pattern Chromatin
• Exhibit very residual RNA explaining the non-basophilia
• Consists of secondary granules (Pinkish to reddish-blue)
• Synthesis of tertiary granules may begin at this stage

Band or Stab

• Elongated, band shaped, markedly indented where indentation is greater than one half the width of the round nucleus
• Dark purple color with
• N/C ratio: 1:1 - 1:2
• Highly clumped, coarse, blue-black pattern Chromatin
• Lacks residual RNA
• Consists primarily of tertiary granules and also secretory granules
• Has normal values in peripheral blood: 1-2%

Monopoiesis

• Monocytic development is similar to neutrophil development because both cell types come from the GMP
• Macrophage Colony-Stimulating Factor (M-CSF) is a major cytokine responsible for monocyte growth and differentiation

Monoblast

• Round or oval, light bluish-purple nucleus
• N/C ratio: 3:1 – 1:1
• Fine and distinct Chromatin pattern
• 1-5 nucleoli
• Light in staining where distinguishing feature from myeloblast
• Lacks granules

Promonocyte

• Oval or slightly indented, light bluish-purple nucleus
• N/C ratio: 2:1 – 1:1
• Fine and uniformed or reticular pattern
• 1-5 nucleoli
• Has occasional vacuoles that become more prominent

Lymphopoiesis

• Each type of lymphocyte follows its own pathway for development, but generally has a similar pattern
• For B cell and T cells, development can be subdivided into antigen-independent and antigen-dependent phases

Lymphoblast

• Round or oval with a central or eccentric reddish-purple nucleus
• N/C ratio: 7:1 – 4:
• fine, lacy to moderately coarse pattern Chromatin
• Have smooth Moderately dark blue cytoplasm and 1 – 2 prominent nucleoli

Prolymphocyte

• Round, centrally placed with a reddish-purple nucleus, abundant cytoplasm and 1 prominent nucleus
• Exhibits a N/C ratio: 4:1 – 3:1
• Has a coarse, clumped pattern Chromatin

B-Lymphocytes

• Develop in the bone marrow through these stages: pro-B cells, pre-B cells, immature B-cells, mature B-cells
• Immunoglobulin gene rearrangement occurs during these stages to produce a unique immunoglobulin antigen receptor
• Immature B-cells migrate to secondary lymphoid organs where they mature and encounter antigens

T-Lymphocytes

• Develop initially in the thymus (cortex)
• Occurs through pro-T cells, pre-T cells, immature T-cells, mature T-cells
• Undergo antigen receptor gene rearrangement to produce unique T-cell receptors
• Subdivide into CD4+ or CD8+ depending on the antigen present on the surface