Hematology: Neutrophils and Genetic Abnormalities

- Leukocytes, or white blood cells (WBCs), get their name from being relatively colorless compared to red blood cells.
- Leukocyte types are identifiable via light microscopy with Romanowsky stain or flow cytometry based on surface antigens.
- Granulocytes are leukocytes with cytoplasm filled with granules, including eosinophils, basophils, and neutrophils.
- Mononuclear cells include monocytes and lymphocytes, characterized by unsegmented nuclei.
- Overall, leukocytes mediate immunity, be it innate (non-specific), like phagocytosis by neutrophils, or specific (adaptive), as in antibody production by lymphocytes and plasma cells.
- Kinetics involves cell movement across developmental stages, into circulation, and from circulation into tissues, including the time spent in each phase of life.

### Hematopoiesis
- Leukocytes originate from hematopoietic stem cells (HSCs) in the bone marrow, where they differentiate and mature before entering circulation.
- The number of circulating leukocytes varies due to factors like sex, age, activity, time of day, ethnicity, stress, consumption, destruction, and bone marrow production.
- Reference intervals for total leukocyte counts vary across labs based on patient population and instrumentation; a typical adult range is 4.5 × 109/L to 11.5 × 109/L.

### Neutrophils
- Neutrophils exist in peripheral blood as either segmented or band forms, with segmented neutrophils predominating in circulation.
- Neutrophils share a common progenitor with monocytes, distinct from eosinophils and basophils, known as the granulocyte-monocyte progenitor (GMP).
- Granulocyte colony-stimulating factor (G-CSF) is the primary cytokine stimulating neutrophil production.
- Bone marrow neutrophil development occurs in three pools, with the stem cell pool consisting of HSCs capable of self-renewal/differentiation.
- The proliferation (mitotic) pool includes dividing cells (CMPs, CFU-GEMMs; GMPs; myeloblasts; promyelocytes; and myelocytes).
- The maturation (storage) pool has cells undergoing nuclear maturation and available for release (metamyelocytes, band neutrophils, and segmented neutrophils).
- HSCs, CMPs, and GMPs are indistinguishable by light microscopy and Romanowsky staining but can be identified via surface antigen detection by flow cytometry.
- Myeloblasts make up 0% to 3% of nucleated marrow cells and measure 14 to 20 µm in diameter; Type I myeloblasts have a high N:C ratio, fine nuclear chromatin, and two to four visible nucleoli.
- Type II myeloblasts display dispersed primary (azurophilic) granules in the cytoplasm (not exceeding 20 per cell).
- Type III myeloblasts, rare in normal bone marrow, contain >20 granules that does not obscure the nucleus, but show darker chromatin and a more purple cytoplasm.
- Promyelocytes make up 1% to 5% of nucleated marrow cells, measure 16 to 25 µm in diameter, possess a round/oval (often eccentric) nucleus; a paranuclear halo ("hof") is commonly seen in normal promyelocytes.
- The cytoplasm is evenly basophilic and filled with primary (azurophilic) granules (the first granule series to be produced during neutrophil maturation).
- Myelocytes make up 6% to 17% of nucleated marrow cells and represents the final stage where cell division (mitosis) occurs; at this stage, primary granule production ceases as the cell begins manufacturing secondary (specific) neutrophil granules.
- This stage is split into early and late myelocytes and secondary neutrophilic granules slowly spread into patches of grainy pale pink cytoplasm in the area of the Golgi apparatus, known as dawn of neutrophilia.
- As the cell divides, primary granule number decreases and their membrane chemistry changes, making them less visible and late myelocytes are somewhat smaller than promyelocytes (15 to 18 µm) as the nucleus has a more heterochromatic appearance.
- Metamyelocytes constitute 3% to 20% of nucleated marrow cells; after this stage, the cells no longer divide and the primary change is the shape of the nucleus: indented (kidney bean-shaped/peanut-shaped), and increasingly clumped chromatin.
- Nucleoli are absent and synthesis of tertiary granules may begin; the metamyelocyte is slightly smaller than the myelocyte (14 to 16 µm).
- Bands make up 9% to 32% of nucleated marrow cells and 0% to 5% of nucleated peripheral blood cells and lack cytoplasmic basophilia; tertiary granules continue to form and secretory granules may begin to form.
- The nucleus is highly clumped and indentation exceeds one-half of the nuclear diameter, and the band counts was thought to be useful in the diagnosis of patients with infection.
- Segmented neutrophils constitute 7% to 30% of nucleated cells in the bone marrow; secretory granules continue to form during this stage and two to five nuclear lobes connected by thread-like filaments is the only morphologic difference between segmented neutrophils and bands.

### Neutrophil Kinetics 
- Once in the peripheral blood, neutrophils randomly divide into a circulating neutrophil pool (CNP) and a marginated neutrophil pool (MNP).
- MNP neutrophils loosely localize to capillary walls in tissues like the liver, spleen, and lung.
- No apparent functional differences exist between CNP and MNP neutrophils, and cells move freely between the two pools.
- The ratio of these two pools is approximately equal overall; the half-life of neutrophils in the blood is relatively short at approximately 7 hours.
- Integrins and selectins significantly allow neutrophils to marginate, exit the blood, and enter tissues through diapedesis.

### Neutrophil Function
-  The cells that do not pass into the tissues go through programmed cell death or apoptosis and macrophages eliminate them in the spleen, bone marrow, and liver.
- The lifespan of polymorphonuclear cells in the tissues is variable upon whether inflammatory agents are responded to and measured in hours.
- Neutrophils are part of the innate immune system that destroys cells in the absence of an antigen.
- Destruction of foreign material/microorganisms through phagocytosis and digestion, seeking (chemotaxis, motility, and diapedesis). Extravasation begins at the inflammatory site when chemotactic agents attach to neutrophil receptors.
- They also roll along vessel walls by using stronger adhesive molecules than nonstimulated ones and also transmigrate either between or endothelial cells in a directional way to the highest density of chemotactic agents.
- Attachment/engulfment come with identification, in which cytoplasmic pseudopodia encircle the particle to produce a phagosome.
- Other functions are generating neutrophil extracellular traps and their secretory aspects
- NETs, or neutrophil extracellular traps, are extracellular thread-like structures that have enzymes that kill pathogens

### Eosinophils
- Eosinophils make up 1% to 3% of bone marrow nucleated cells and in peripheral blood, with an absolute number of up to 0.4 × 109/L.
- Promyelocytes identified using Charcot-Leyden crystal protein in primary granules; maturation begins with the early myelocyte.
- Electron micrographs show many secondary granules with an electron-dense crystalline core and resemble their neutrophil counterparts, generating secretory granules.
- Mature eosinophils have a bilobed nucleus; the cytoplasm has refractile granules and their number grows when the eosinophil gets activated
- The time after late myelocyte is 3.5 days and their mean turnover is 2.2 x 108 cells/kg daily; Eosinophil granules also contain cytokines, chemokines, and cationic proteins.
- Granules move towards plasmatic membrane by way of many forms of exocytosis.
- Eosinophils involved in cell killing are also able to act as promoting effectors. Transmigrate to thymus for newborn deletion in thymocytes.
- They regulate mast cell function through MBP, cause cytokine production, and give nerve growth factor.
- Production boosted through parasitic helminths and allergy and causes some forms of damage.

### Basophils
- They are derived from progenitors and differentiate under many cytokines including IL-3 and TSLP.
- Populations characterized by the responses these elicit.
- Immature ones have round to somewhat lobulated nuclei with only slightly condensed chromatin, blue cytoplasm, and large blue-black secondary granules.
- Primary azure granules may or may not be present.
- Granules are water soluble and may dissolve if the blood film is washed too much during staining.
- Mature basophils contain a lobulated nucleus often obscured by granules and when visible is clumped and is usually about 60 hours.
- Actual nuclear segmentation with visible filaments occurs rarely and the cytoplasm is colorless with high amounts of large blue-black granules.
- These least numerous WBCs in blood and slightly condense.
- Their kinetics are poorly understood due to few amounts and has higher activation with cell death.
- Basophil functions such as acting to release multiple subtypes of large quantities are not well known and can synthesize granule protein.
- Are found in innate and adaptive immunity
- Cells for inflammation and mediators of other allergies.

### Mast Cells
- Have MCPs that originate from stem bone and spleen
- The MCPs also function as antigen-presenting cells and act as mediators in both innate and adaptive immunity

### Monocytes
- Monocytes makeup 2% of total WBC
- Development just like neutrophils including being derived from GMP
- Once in tissues, they differentiate in osteoclasts and other cell types to immune functions
- Pattern recognition receptors can produce nitric which is known as monocytes/macrophages
- Have oval nucleus, and some are known as resident and also dendric

### Lymphocytes
- Divided into three major cells that can be allogenic and create immunitary response having CD4+CD25+ regulatory T cells
- Have resting and self renewal ability to respond to antigens
- Develop and mature from the bone marrow and thymus for T and NK and the development into dependent antigen phases to those two cells for allogenic cells
- It is in the auxiliary or secondary organs where B cells can get into connection and results in cell division, 7% to 30%
- In some conditions , B cells may have help produce optimal CD4 activation

### Plasma cells
- Cell division can involve T and B antigens and can be an example of cell action
- Helper and helper cell types for the main response
- Cytotoxic cells target killing that secrete cell death or inhibiting