Pharmacy Program

Delivering Integrated Care Management

The IHTC pharmacy and healthcare professionals interact on a daily basis at our center to maximize coordination and quality of care. Our pharmacists and physicians are on call and available 24 hours a day, seven days a week.

The IHTC pharmacy and healthcare professionals effectively coordinate ongoing care by proactively communicating with our patients to manage clotting factor needs, therapy compliance, and bleeding episodes.

White Blood Cell Disorders

  1. Overview
  2. Hematopoiesis
  3. Structure and Function of the Granulocytes
  4. Structure and Function of the Monocytes
  5. Phagocytosis and the Respiratory Burst
  6. Structure and Function of the Lymphocytes
  7. Age Dependent Values of Leukocytes
  8. Pathological Alterations of Leukocyte Number
  9. Clinical Approach to a Child with Neutropenia
  10. Laboratory Evaluation for a Child with Neutropenia
  11. Evaluation of the Patient with Recurrent Infections
  12. References


White blood cells (leukocytes) orchestrate the host response to pathogens. Leukocytes are divided into myeloid (eosinophils, monocytes, neutrophils, basophils) and lymphoid (B cells, T cells, and natural killer (NK) cells) elements. Monocytes and neutrophils mediate innate immune defenses and present antigens to lymphocytes, the principal effectors of the adaptive immune system. Eosinophils and basophils defend against parasitic pathogens, play important roles in inflammation, and mediate allergic reactions.

Monocytes and neutrophils, as part of innate immunity, phagocytose bacteria and debris, eliminate virus-infected cells, and destroy parasites and fungi. Phagocytosis is assisted by serum complement proteins, which adhere to pathogens and promote chemotaxis and opsonization. Monocytes mature in tissue to become macrophages, where they, along with dendritic cells and B cells, serve as antigen presenting cells (APCs) to the adaptive immune system.

B and T cells, as part of adaptive immunity, confer long-term protection against pathogens, especially extracellular bacterial infections. Adaptive immunity orchestrates both antibody-mediated humoral immunity and T-cell mediated cellular cytotoxicity. B cells secrete neutralizing antigen-specific antibodies (i.e. humoral) upon stimulation by the macrophages, dendritic cells, and T cells. In contrast, T cells and NK cells, upon stimulation, can directly kill aberrant cells (i.e. cell-mediated). These aberrant cells include those infected with viruses, bacteria, and fungi, tumorigenic cells, and transplanted tissue.


Leukocytes arise from pluripotent hematopoietic stem cells in the bone marrow (See figure: Hematopoiesis). Under the influence of cytokines IL-6, SCF, and Flt3L, these stem cells develop into common myeloid (i.e. granulocytes and monocytes) or common lymphoid (i.e. lymphocytes) progenitors. Growth factors such as granulocyte-macrophage-colony stimulating factor (GM-CSF) and interleukin-5 (IL-5) promote the further differentiation of myeloid and lymphoid progenitors into terminally differentiated forms.

Myeloid progenitor stem cells differentiate in the bone marrow to yield committed cell types called colony forming units (CFU). These CFUs eventually give rise to terminally differentiated leukocytes. Eosinophils develop in a distinct lineage through IL-5 signaling. Megakaryocytes, erythrocytes, and basophils develop in a common lineage through thrombopoietin,  IL-11, and IL-3 signaling, while monocytes and neutrophils develop in a common lineage through GM-CSF, G-CSF, and M-CSF signaling. Monocytes mature in tissue to become macrophages, while neutrophils primarily circulate in the blood. In neutropenic or infected states, activated macrophages increase neutrophil production by releasing IL-1, IL-6, tumor necrosis factor, and other cytokines which induce stromal T cells to produce colony stimulating factors.

Lymphoid progenitor stem cells differentiate through SCF, Flt3L, and IL-7 signaling to become Pro-B, Pro-NK, and Pro-T cells. Pro-B cells and Pro-NK cells mature in the bone marrow to become B cells and NK cells, while Pro-T cells mature in the thymus through IL-7 signaling to become T cells. B cells undergo further maturation in the bone marrow and in the lymphatic tissue to become plasma cells. Plasma cells actively secrete antigen-specific antibodies.

Structure and Function of the Granulocytes


Eosinophils circulate in the peripheral blood and compose only a small fraction of nucleated bone marrow cells (0.3%) o 8%).e in the bone marrow from the CFU-Eo progenitor under IL-5 signaling. Mature eosinophils measure about 15 um, have a bilobed nucleus, and stain orange-pink (i.e. eosinphilic) due to distinct granules containing basic proteins.

Eosinophils extravasate primarily to environmentally exposed tissues (e.g. GI tract, subcutaneous tissue) and mediate allergic reactions, tumor defense, and parasitic killing, especially against helminthes. Eosinophils, like other phagocytes, respond to and are activated by protein ligands, immunoglobulins, complement protein fragments, and chemotactic molecules.

Eosinophilic granules measure approximately 1 um2 and contain major basic protein, arylsulfatase, hydrolytic enzymes, cathepsin, and peroxidase. Eosiniphilic peroxidase generates hypobromous acid from hydrogen peroxide, and major basic protein disrupts membranes and DNA, substances important in  parasite defense. Additonally, the eosinophil (and basophil) plasma membrane contain lysophospholipases, which polymerize to form the Charcot-Leyden crystal, an important histological marker for sites of allergy and parasitic infection.

Eosinophils also suppress inflammatory reactions. The eosinophil chemotactic factor of anaphylaxis, a substance released by mast cell and basophil degranulation, attracts eosinophils to sites of hypersensitivity inflammation. Eosinophils secrete histaminases, phospholipase B, major basic protein, and lysophospholipase, which serve to inactivate histamine, PAF (platelet-activating factor), heparin, and arachidonic acid metabolism, respectively.


Basophils compose 0.5% oells and 0 to 3% odifferentiate under IL-3 signaling in a lineage shared with megakaryocytes and red blood and mature in the bone marrow within 7 days. Mature basophils measure about 12 um, have a bilobed nucleus, and stain dark blue due to granules containing sulfated glycosaminoglycans, including histamine, heparin, and chondroitin sulfate. 

Basophils express the IgE Fc region receptor, and activated basophils are labeled with antigen-specific IgE molecules. Upon antigen binding, signal transduction induces degranulation, releasing inflammatory molecules such as histamine and leukotriene C4. These molecules induce mucus secretion, smooth muscle contraction, and vasodilation, producing the characteristic symptoms associated with hypersensitivity reactions.

While basophils and tissue mast cells share many structural and functional characteristics, they are not equivalent cells. Mast cells do not circulate in the blood and are suspected to arise from distinct bone marrow progenitors.


The polymorphonuclear leukocytes (PMNs) circulate in the peripheral blood and are the most numerous of white blood cell types, comprising 40 to 70% oirculating neutrophil number represents approximately 5% oss, which principally remains in the bone marrow. Neutrophils differentiate in the marrow under GM-CSF and G-CSF signaling and mature from myeloblasts through band forms to neutrophils in approximately 14 days. Mature neutrophils are 10 to 15 um with a multi-lobed polymorphic nucleus and yellowish granule-containing cytoplasm.

In response to G-protein coupled chemotactic signals from molecules such as bacterial peptides, complement protein fragments C3a and C5a, and chemokines (e.g. IL-8, GRO peptides), neutrophils leave the blood and enter tissues to perform phagocytic functions. This honing process occurs in several stages and is the basic mechanism by which all leukocytes – not just neutrophils – enter the tissue:

  1. Random contact with the endothelium.
  2. Rolling along the endothelium, mediated primarily by selectin protein binding to fucosylated carbohydrates on the endothelium.
  3. Adhesion to the endothelium, mediated by integrin protein (e.g. LFA-1) binding to intracellular adhesion molecules (ICAMs) on the endothelium (see Leukocyte Adhesion Deficiency).
  4. Diapedesis, or the penetration of endothelial tight junctions and basement membrane by migrating leukocytes, effected through increased calcium concentrations as a result of ICAM signaling.
  5. Chemotaxis of the leukocyte via chemoattractant molecule concentration gradients. In neutrophils, chemotaxis is directed by cytoskeletal F-actin containing pseudopodia and lamellepodia.

One at the site of infection or inflammation, neutrophils kill microbes and phagocytose inflammatory debris, functioning for 1-2 days before being phagocytosed themselves by macrophages.

Neutrophilic killing occurs through phagocytosis and the release of digestive enzymes and substances contained in cytoplasmic granules. Azurophilic (primary) granules are peroxidase positive, contain cysteine-rich defensin peptides, permeability-increasing proteins, and serine proteases. Specific (secondary) granules contain lactoferrin, an iron-sequestering protein, cytochrome-b558, a molecule critical for the neutrophil’s respiratory burst, and a reserve pool of chemoctactic receptor proteins. Tertiary granules contain gelatinase, a matrix metalloproteinase.

Neutrophil activation, phagocytosis, and degranulation proceed through a variety of ligand, cytokine, and chemoattractant based extracellular signaling mechanisms, including G-protein linked and tyrosine kinase receptor activity. Importantly, G-protein signaling results in increased intracellular calcium levels, which induces degranulation through phagolysosome fusion. Downstream intracellular effectors include the Ras and Rac GTPases, molecules which regulate chemotaxis and the respiratory burst NADPH oxidase, respectively. 

Structure and Function of the Monocytes

Monocytes compose 4 to 11% odifferentiate under GM-CSF and M-CSF signaling in a lineage shared with neutrophils. They mature within one day and enter the circulation. Circulating monocytes measure approximately 15 um, show an irregular membrane, have a kidney-shaped nucleus, and contain a singular type of lysosomal granules.

Monocytes circulate briefly in the blood before extravasating into tissue as macrophages, where they live up to three months. Monocytes etxravasate in a process similar to that described for neutrophils; important chemokines for monocytes include MCP-1 (monocyte chemoattractant protein-1) and RANTES (regulated upon activation, normal T cell expressed and presumably secreted). Morphologically, macrophages differ from monocytes in that they show an oval nucleus with prominent nucleoli and more basophilic cytoplasm, features that indicate increased RNA synthesis.

Multiple cytokines, toxins, and local tissue signals activate macrophages, but interferon gamma (IFN-γ), released by T lymphocytes, neutrophils, and macrophages is the principal macrophage activating molecule. IFN-γ binds to its extracellular receptor and signals through the intracellular Jak-STAT pathway (Janus kinase-signal transducers and activators of transcription pathway), modulating transcription of vital macrophage activating genes. Likewise, bacterial lipopolysaccharide (endotoxin) and local tissue signals can activate macrophages.

Macrophages kill intracellular parasites (e.g. Listeria, Mycobacterium, some fungi), induce humoral and cellular immunity via interactions with T and B cells, and exhibit anti-tumor activity. Macrophages play additional roles in wound repair, tissue remodeling, inflammation, hematopoiesis, and the scavenging of low density lipoproteins (LDL). To modulate these diverse functions, macrophages live in multiple tissue compartments and produce a host of effector molecules, including digestive enzymes, oxidants, binding proteins (e.g. avidin), coagulation factors, arachidonic acid metabolites, complement proteins, cytokines (including TNFα and the CSFs), angiogenic, fibroblast, and epithelial growth factors, matrix proteins (e.g. fibronectin), and hormones such as 1α, 25-dihydroxyvitamin D3.

Macrophages are distributed throughout the body. Splenic macrophages clear debris from the red pulp and sinus and stimulate lymphocytes. Macrophages in the lymph nodes ingest, process, and present antigens to T lymphocytes for activation of the adaptive immune system. Kupffer cells in the liver sample the hepatic blood that filters through the space of Disse into the sinusoids, and pulmonary macrophages (dust cells) phagocytose inhaled debris and microorganisms. Scavenging macrophages additionally reside in the GI tract and mammary glands. In the bone marrow, macrophages release hematopoietic factors and phagocytose defective maturation products.

Multinucleated osteoclasts resemble macrophages in surface receptor profiles and have been shown to arise from GM-CFUs in transplantation studies. Osteoclasts reabsorb bone, a process augmented by the administration of IFN-γ. Finally, macrophage-like cells reside in the lymph fluid (veiled cells) and in the epidermis (Langerhans cells). Despite developing separately from the monocytic macrophages, Langerhans cells and veiled cells play important roles in antigen processing and presentation.

Phagocytosis and the Respiratory Burst

Phagocytosis (from the Greek phago, to eat) is the ingestion and processing of microbes, cell remnants, and other debris, orchestrated by macrophages and recruited neutrophils. Opsonins (from the Greek opsonein, to prepare for dining), including serum complement proteins and secreted serum immunoglobulins, allow macrophages and neutrophils to recognize and phagocytose pathogens. Tissue macrophages also possess opsonin-independent mechanisms of phagocytosis; for example, the macrophage mannose binding protein (MBP) receptor recognizes bound mannose, a carbohydrate component of many bacterial, viral, protozoal, and fungal surfaces. Deficiencies in antibody production and complement proteins can result in increased susceptibility to bacterial infection.

Phagocytosis occurs in the following general steps:

  1. Opsonization: opsonic antibodies IgM and IgG and complement C3b and C3b label pathogens for killing.
  2. Opsonin binding: phagocytic cells bind to complement or antibody molecules through their complement receptor (e.g. CR1 and CR3) or Fc receptor (e.g. FcγR, the IgG constant domain receptor).
  3. Phagocytosis: receptor binding induces membrane invagination and pseudopodia extension through actin cytoskeletal rearrangement, forming a phagocytic vacuole called a phagosome.
  4. Killing: granule plasma membranes fuse with the phagosome plasma membrane, releasing cytotoxic substances into the newly formed phagolysosome. Simultaneously, the release of pre-formed receptor molecules, such as integrins, stored in the granules enhances the immune response. Pathogens such as Salmonella and Listeria monocytogenes evade death by interfering with the fusion and acidification of the phagolysosome.

The Respiratory Burst

Killing depends on both oxygen-independent mechanisms (e.g. pre-formed digestive proteins) and the oxygen-dependent respiratory burst. The respiratory burst increases a neutrophil’s oxygen demand 100-fold, a demand compensated for by vasodilation and fever. The respiratory burst utilizes NADPH oxidase, a four polypeptide enzyme complex composed of membrane-bound and soluble elements (see Myeloperoxidase Deficiency; see Chronic Granulomatous Disease).

Upon activation by chemoattractant, Fc, or complement receptor binding, NADPH oxidase converts elemental oxygen (O2) to superoxide (O2-). Superoxide converts superoxide to hydrogen peroxide (H2O2). A small amount of superoxide and hydrogen peroxide non-enzymatically react with ferrous iron to produce hydroxyl free radicals (OH). While catalase and glutathione peroxidase convert some of this hydrogen peroxide to oxygen and water, myeloperoxidase converts hydrogen peroxide to hypochlorous acid (HOCl), an antimicrobial molecule that is also the active ingredient in household bleach. Myeloperoxidase, along with hydrogen peroxide and the hydroxyl radical, participate in microbial killing within the phagolysosome. All three molecules serve as potent activators of metalloproteinases and inactivator of antiproteinases, allowing phagocytes to break down the extracellular matrix in order to migrate through inflamed tissue.

(Figure: The Respiratory Burst)


Lymphocytes mediate adaptive immunity and include T cells, B cells, and NK cells. T cells activate adaptive immune responses after encountering pathogenic molecules presented by professional antigen presenting cells (APCs), which include macrophages, dendritic cells, and B cells. B cells differentiate into immunoglobulin-producing plasma cells or into memory B cells, which stimulate faster adaptive immune responses when a pathogen is re-encountered. The principal function of each lymphocyte is as follows.

  • T cells: adaptive immune responses (CD4 ), cell-mediated immunity (CD8 ).
  • B cells: antigen presentation,  immunoglobulin production, , immunological memory.
  • NK cells: directly cytotoxic, especially toward virus-infected and tumor cells.


Lymphocytes comprise 22 to 44% olood cell population. They measure from 6-10 um (similar to erythrocytes), show ovoid nuclei, dense chromatin, and sparse cytoplasm. NK cells can be larger, measuring up to 15 um, and contain granules composed of granzyme and perforin, proteins which NK cells use to kill virus-infected cells.

T lymphocytes

T cells comprise 60 to 70% oion of lymphocytes. T cells mature in the thymus, where their T cell receptor (TCR) genes undergo somatic rearrangement and the mature T cell becomes either CD4 or CD8 . This rearrangement produces antigen-specificity; i.e. each T cell’s TCR has a unique structure and antigen-recognition capability. The TCR recognizes only protein. In contrast B cells can recognize and be activated by protein, polysaccharides, glycolipids, and nucleic acids. Mature T cells are always CD3 and ζ , critical transducer molecules of extracellular signals and also important markers used in cytometric analysis of leukemia and lymphoma. T cells are additionally classified according to structural and functional differences.

  • CD4 T cells (60% oh their CD3 , ζ , and CD4 T-cell receptor (TCR), bind to the class II major histocompatibility complex (MHC) found on professional antigen presenting cells (macrophages, B cells, and dendritic cells).
    • CD4 TH1 cells.
      • Via IL-2 and IFN-γ signaling, mediate delayed hypersensitivity reactions, activate macrophages, and upregulate IgG synthesis.
      • Important in intracellular parasite defense (e.g. virus, fungus, some bacteria).
    • CD4 TH2 cells.
      • Via IL-5, IL-5, and IL-13 signaling, upregulate IgE synthesis and activate eosinophils.
      • Important in helminth defense and allergic reactions.
  • CD8 T cells (30% oThrough their  CD3 , ζ , and CD8 TCR, bind to class I MHC molecules, which are found on nucleated cells throughout the body (professional and non-professional antigen-presenting cells).

T cell activation require two signals. TCR binding to the MHC-antigen molecule provides the first signal. The second signal  comes from the binding of CD28 on the T cell with CD80 or CD86 on the antigen-presenting cell. Without the CD28:CD80/86 binding, T cells do not become activated. Once activated, T cells produce large amounts of IL-2, which acts in autocrine and paracrine fashion to promote antigen-specific proliferation and the differentiation of memory T cells.

B lymphocytes

Lymphoid progenitors give rise to Pre and  Pro-B cells, which undergo a series of ordered Ig gene rearrangements to become circulating Mature B cells. Mature B cells comprise 10% tlation of lymphocytes.

  • Pro-B cells, Pre-B cells, and Immature B cells.
    • Defined by progressive stages of immunoglobulin gene rearrangement and expression.
    • Employ unique genetic strategies to generate immense immunoglobulin diversity.
    • Have undergone negative selection. Only self non-reactive B cells mature.
  • Mature B cells.
    • Leave the bone marrow to enter the circulation.
    • Express antigen-specific surface IgM and IgD.
      • Activate intracellular signal transduction upon antigen binding.
      • Internalize bound antigen and present to T cells.
    • Undergo both T cell-dependent (specialized) and T cell-independent responses (generalized).
  • Plasma cells.
    • Secrete opsonizing antibodies IgG1-4, IgA, or IgM.
    • Exhibit high nucleus:cytoplasm ratio and characteristic “clockface” chromatin.


NK Cells

Natural Killer cells, which are structurally comparable to yet unique from T cells, comprise 10-15% oion of lymphocytes.  NK cells were originally thought to recognize and target cells that demonstrate a low level of MHC class I expression, which certain intracellular pathogens have evolved to downregulate.  However, recent research demonstrates that NK cells recognize not only the lowered expression of class I molecules, but also MHC-like and non-MHC molecules. 

NK cells are CD56 , NKp46 , CD3-, and CD16 or CD16-. They are thought to originate in the bone marrows, travel to the lymph nodes via the high endothelial venule into the parafollicular space where they undergo further maturation, and return to the circulation via efferent lymph.

NK cells function to both upregulate the immune response via cytokine release (principally IFN-γ) and to effect cell death though the secretion of perforin/granzyme and through the death receptor pathways (e.g. Fas and TRAIL). NK cells  possess two inhibitory receptors (killer immunoglobulin-like receptors, or KIR and CD94-NKG2A) that recognize and bind MHC class I molecules, preventing the NK cell from inducing apoptosis. In the absence of MHC class I molecules and in the presence of activating signals, NK cells induce apoptosis.  The NK cell activating receptors are still being elucidated. The best known is NKG2D, a C-type lectin-like receptor which recognizes at least 6 ligands (transmembrane proteins and glycophosphatidylinositol anchored proteins), all of which are expressed only when the host cell is under viral or tumorigenic stress. (from Caligiuri, Blood, 2008).

Age-Dependent Values for Leukocyte Number

Leukocyte numbers vary according to age. Understanding and awareness of age appropriate norms of leukocyte numbers is crucial to interpret disorders of leukocytosis and leukopenia. Table “Leukocyte Counts by Age” and Figure “Age-Dependent Leukocyte Values” illustrates the age-appropriate ranges for the various subsets of leukocytes.

Pathological alterations of leukocyte number

Neutropenia and Neutrophilia

Neutropenia describes a low neutrophil number in the peripheral blood and is defined as <1500 cells per mcL of blood. Neutropenia may be a result of either decreased production in the marrow or increased peripheral destruction. Neutrophilia describes a high neutrophil number and is usually secondary a non-hematological disorder. Causes of neutrophilia include increased production (e.g. infection), enhanced release from the marrow reserve, decreased removal from the blood (e.g. post-splenectomy), and as a result of reduced margination (e.g. during epinephrine administration). (Causes of Neutropenia, Neutrophilia, and Eosinophilia)

Clinical Approach to a Child with Neutropenia

Laboratory Evaluation for a Child with Neutropenia

Coming soon!

Evaluation of Recurrent Infections


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Kumar V, et al [eds]. Robbins and Cotran Pathologic Basis of Disease, 7th ed. Philadelphia, WB Saunders, 2005, 193-264; 620-622; 661-666.

Kratz A, et al. Laboratory Reference Values. N Engl J Med 2004;351:1548-63.

Dinauer MC. The phagocyte system and disorders of granulopoiesis and granulocyte function. In Nathan DG and Orkin S [eds.]. Nathan and Oski’s Hematology of Infancy and Childhood, 5th ed. Philadelphia, WB Saunders, 1998; 889-967.

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