Hematology 425, Leukopoiesis

04/13/231

Hematology 425 Hematology 425 LeukopoiesisLeukopoiesis

Russ MorrisonOctober 11, 2006

04/13/232

LeukopoiesisLeukopoiesis

Leukopoiesis is the development of WBCs

WBC development (except lymphycytes) occurs in the same locations as RBCs (review figure 6-1)

In WBCs the maturation changes are more unidirectional since with the exception of neoplasia or myeloid metaplasia, the spleen and liver do not participate in WBC formation after birth

04/13/233


Though one term, erythron, is used to define RBC production, there is no corollary for WBC production

WBC production involves complex populations of cells with different compartments that they occupy during their life cycle

Control mechanisms of cellular behavior are more complex in WBCs than in RBCs

04/13/234


WBC control mechanisms include interrelations with adipose tissue, fibroblasts and endothelial cells

Again, as with RBCs, cellular production takes place in all marrow space at birth and by the end of adolescence is found only in the marrow of the proximal ends of the long bones and in flat bones such as the skull and sternum. (review fig.6-2)

The inactive marrow of adolescence and adulthood can revert to active marrow in times of stress

04/13/235


WBCs can be divided into categories based on specific function, site of origin or morphology

All WBCs exist to defend the body against “nonself” agents

This defense is accomplished through intricate cooperation and communication among cells

04/13/236


As a for instance, phagocytes attack and destroy a wide variety of invading matter on their own. However, lymphocytes direct and amplify phagocytic action through the release of lymphokines (a sort of bioresponse mediator)

WBCs are divided into granulocytes and lymphocytes based on differentiation at the primitive stem cell level (fig. 11-1)

04/13/237


Lymphocytes are produced in both bone marrow and lymphoid tissue

Environmental and hormonal stimuli of lymphocytes are different than those that control granulocytes and monocytes

Granulocytes (PMNs) function as destroyers of pyogenic bacteria, monocyte/macrophages are less descriminating in their dietary preferences

04/13/238


Granulocytes contain visible granules and develop in the bone marrow

Granulocytes are subdivided according to morphology and according to size/visibility of granules

Cells containing large, visible granules are called granulocytes and are further divided into PMNs, Eos and Basos based on differential staining of the granules with Romanowsky-based stain

Monocytes contain tiny granules that cause their cytoplasm to appear grainy with light microscopy

04/13/239


Microscopic evaluation of WBCs is the basis of clinical study

Flow cytometry of receptor sites, antigenic labeling and even functional studies now contribute to clinical information gathered in the diagnosis and management of disease

Cell markers have been given alphanumerical codes (CD1) in which CD stands for cluster designation, discussed in chapter on flow cytometry

04/13/2310

Leukopoiesis, GranulocytesLeukopoiesis, Granulocytes

Found in high concentrations in 4 locations called granulocyte pools

1. Bone marrow2. PB circulation3. Marginating up against the endothelium

of blood vessels4. Tissues

04/13/2311


Bone marrow pool is large and has 3 functions

1. Proliferation2. Maturation3. Storage Cells found in the proliferating

component are myeloblasts, promyelocytes and myelocytes, all capable of mitotic division

04/13/2312


The maturation component of the BM consists of metamyelocytes and band forms no longer capable of mitosis but not yet fully functional

The storage component of the BM consists of bands and PMNs and holds 25x as many cells as in the circulating PB

04/13/2313


Fully mature granulocytes are stimulated by chemotactic factors and leave the marrow entering the PB where they become part of either the marginating pool of the circulating pool

The marginating pool consists of 50% of total PB granulocyte levels where the cells have adhered to blood vessel endothelium or are engaged in diapedesis (egressing into tissue through vessel walls)

04/13/2314


The circulating pool contains the remaining 50% of PB granulocytes and are the cells seen and counted in PB hematologic studies

Granulocytes move freely between marginating and circulating pools in a bi-directional flow for a variety of reasons

04/13/2315


Maturation of the Granulocytic SeriesBegins with the pluripotential stem cell

(PSC)PSC commits its progeny to lymphoid or

bone marrow origin, for reasons unknown, through the action of growth factors that are either tyrosine kinase receptors or cytokines

04/13/2316


For granulopoiesis, the PSC undergoes stimulation, mitosis and maturation into a stem cell that is specific for bone marrow-derived or myeloid cells

This CFU-GEMM matures into another stem cell called the CFU-GM

The CFU-GM matures into the earliest cell of the neutrophilic series, the myeloblast

04/13/2317


Cell numbers and function is controlled by complex interaction of humoral factors such as interleukins and CSFs

CSFs are categorized by the type of cell stimulated

GM-CSF – granulocytes & monocytes/ macrophages

G-CSF – granulocytesM-CSF - monocytes/macrophages

04/13/2318


CSF specificity is mediated by receptor sites on precursors and on mature cells

Biologic action of receptors consists of a ligand specific low-affinity binding chain and a second, high-affinity chain for binding and signal transduction.

The second chain interacts with IL-3 and IL-5 (Chapter 6)

04/13/2319

Neutrophil Maturation - MyeloblastNeutrophil Maturation - Myeloblast

Cells in the BM proliferation pool take 24-48 hours for a single cell cycle

Less than 1% of the normal BM compartment is composed of myeloblasts

Large, 15-20 um in sizeDelicate nucleus with prominent nucleoliSmall amount of cytoplasm with rough

endoplasmic reticulum, a developing Golgi apparatus and an increasing number of azurophilic granules

04/13/2320

Neutrophil Maturation - MyeloblastNeutrophil Maturation - MyeloblastCytochemical staining shows presence of

myeloperoxidase which is required for intracellular kills

Killing function is the first to be operational in the neutrophil cell line

Myeloblast is incapable of motility, adhesion and phagocytosis and is therefore nonfunctional

04/13/2321

Neutrophil Maturation - Neutrophil Maturation - PromyelocytePromyelocyteAfter a few days in the blast stage, the cell

becomes a promyelocyte1-5% of BM compartment composed of

promyelocytesSize is variable and may exceed 20 um, so

may be larger than myeloblastNuclear chromatin may be delicate or may

show slight clumpingNuceloli begin to fade

04/13/2322

Neutrophil Maturation - Neutrophil Maturation - PromyelocytePromyelocyteGranules are present throughout the

cytoplasm and on top of the nucleusMotility may develop by the end of this

stageMyeloperoxidase is found throughout the

cell which with other enzymes can provide the peroxidase/superoxide burst capable of intracellular kill

04/13/2323

Neutrophil Maturation - MyelocyteNeutrophil Maturation - Myelocyte

Production and accumulation of neutrophilic granules is characteristic of the myelocyte

The myelocyte is the last cell of the BM compartment capable of mitosis

Myelocytes demonstrate morphologic variability as this development stage lasts from 4-5 days and cause alterations in the staining characteristics of the cell

04/13/2324


Smaller in size than the promyelocyte (12-18 um)

Less than 10% of BM compartment is made up of myelocytes

Nucleus is round to oval with a flattened side near the now well-developed Golgi apparatus

Nuclear chromatin shows clumpingNucleoli no longer visible

04/13/2325


Secondary granules stain pink causing a “dawn of neutrophilia” or pink blush within the cytoplasm

Compounds such as alkaline phosphatase begin to concentrate in the cell

The cell acquires some motility

04/13/2326

Neutrophilic Maturation - Neutrophilic Maturation - MetamyelocyteMetamyelocyte The myelocyte becomes a metamyelocyte with

the cessation of all DNA synthesis Delineator of maturation change is that the

nucleus of the metamyelocyte becomes indented with clumped chromatin

Complete collection of primary and secondary granules used to kill and degrade toxic, infectious or non-self agents

Cell is not yet capable of responding to chemotactic factors or of initiating phagocytosis

04/13/2327

Neutrophilic Maturation - Neutrophilic Maturation - MetamyelocyteMetamyelocyte13-22 % of BM compartment10-15 um in sizeNot seen in normal PBNot fully functional, part of the maturation

component of the marrow

04/13/2328

Neutrophilic Maturation - BandNeutrophilic Maturation - Band

The band is a transitional form that exists in both the PB and the BM and considered part of both the maturation and storage pools

Up to 40% of the WBCs of the BM are bands

Represents the “almost mature” neutrophil having full motility, active adhesion properties, and some phagocytic ability

04/13/2329

Neutrophilic Maturation - BandNeutrophilic Maturation - Band

Band forms begin to produce tertiary granules

Membrane maturity shows changes in cytoskeleton, surface charge and presence of receptors for complement

Once entered into the PB, account for less than 6% of circulating WBCs

10-15 um in sizeFound in marginating and circulating poos

of the PB

04/13/2330

Neutrophilic Maturation - PMNNeutrophilic Maturation - PMN

This cell’s nucleus continues to indent until thin strands of membrane and heterochromatin form into segments, hence it is also called a “seg”

Polymorphonuclear means “many-shaped nucleus”, describing the varied nuclear shapes

Cell is completely functional and spend time in the storage pool of the BM as well as marginating and circulating pools of the PB

50-70% of circulating WBCs of PB

04/13/2331

Neutrophilic Maturation - PMNNeutrophilic Maturation - PMN

PMNs spend their life performing phagocytosis and pinocytosis

Phagocytosis involves larger material and can be observed with light microscopy, pinocytosis involves small material (liquids) and is observed with EM

Both of these function can be performed in the circulation of the blood stream or in the tissues

04/13/2332

Eosinophil MaturationEosinophil Maturation

Close relative of the PMN whose secondary granules stain orange-red with Romanowsky-based stains

Development of PSCs into eosinophils requires IL-3, IL-5 and GM-CSF and is inhibited by the presence of interferon

CFU-GEMM to CFU-Eo to myeloblastMyeloblast to promyelocyte which is

indistinguishable from other promyelocytes

04/13/2333


Myelocyte becomes distinguishable from neutrophilic line due to presence of large, round granules containing major basic protein, which in turn is responsible for the staining qualities of the eosiniphilic granules.

Eosinophils spend less than 1 week in the PB

Large storage capacity of Eos in BM allow rapid deployment, on demand

04/13/2334


When stimulated, Eos leave the marrow and pass quickly into the tissues

Actively motile, using same migration paths as neutrophils

Short transit times in PB cause variability in Eo numbers in the WBC differential

Less than 5% of circulating WBCsAllergic response may increase numbers of

Eos

04/13/2335


Mature Eos may be in band form or bilobed while nuclei with higher lobe counts are seldom seen

Slightly larger than PMN at 12-17 um

04/13/2336

Basophil MaturationBasophil Maturation

Characterized by presence of large, purple granules

Granules are irregularly shaped, unevely distributed and deep purple to black when stained with Romanowsky stains

Maturation from stem cell to mature Baso is not well defined, but thought to parallel that of the Eo

04/13/2337

Basophil MaturationBasophil Maturation

As with Eos, Basos can be classifed as myelocytes, metamyelocytes, bands and PMN cells on the basis of nuclear development

As with Eos, mature cells with more than 2 nuclear lobes are not usually seen

The least common cell in the PB, at less than 1% of circulating WBCs

Have high-affinity receptors for the Fc region of IgE

04/13/2338

Monocyte/Macrophage MaturationMonocyte/Macrophage Maturation

Monocyte/Macrophage cells mature from monoblast to promonocyte to blood monocyte to free and fixed macrophages, but the mechanism of commitment is not well understood.

Granular content vary considerably with more than 50 secretory compounds having been dentified.

PB monocytes demonstrate morphologic variability

Aggressive motility and adherence may distort the monocytes during PB smear preparation

04/13/2339


Monocyte nucleus is indented or curved with chromatin that is lacy with small clumps

Typically the largest cell in the PBCytoplasm is filled with minute granules

that produce a cloudy appearanceCytoplasmic membrane may be irregular,

pseudopods and phagocytic vacuoles may be evident

04/13/2340


Described as a transitional cell because it leaves the BM to enter the PB and then leaves to enter tissues in response to chemotactic factors

Makes up les than 15% of PB WBC differential Highly motile and tend to marginate along vessel

walls with a strong tendency to adhere to surfaces

May be stimulated to undergo diapedesis and become free macrophages with increased phagocytic activity

04/13/2341


Macrophages are large, acively phagocytic cells with a size of 15-85 um

Pleomorphic in shape, frequently with pseudopods

Function is phagocytosisMaterial ingested is highly variablePinocytosis also occurs with items less

than 2 um in size

04/13/2342


Multistep process of recognition/ attachment, ingestion, intracellular kill, digestion/degradation, and exocytosis occurs in both phagocytosis and pinocytosis.

Monocytes kill any recognizable non-self agents including dead or dying cells, bacteria, fungi and viruses.

Play a role in processing antigens for lymphocyte recognition and stimulation of lymphocyte transformation.

04/13/2343


May function as anti-tumor agents by phagocytic action of nonself cells via elaboration of tumor necrosis factor and stimulation of lymphocyte activity

Macrophages are in 2 categories1. Free – found in varying concentrations in

all sites of inflammation and repair, alveolar spaces and peritoneal and synovial fluids

04/13/2344


2. Fixed – found in specific concentrations in specific sites such as the nervous system (microglial cells), liver (Kupffer cells), spleen, bone marrow and lymph nodes

Macrophages are large, 15-80 um, have ample cytoplasm filled with granules and often have multiple vacuoles

Nucleus is round to reniform and may contain 1 or 2 nucleoli

04/13/2345

LymphocytesLymphocytes

The only human WBCs whose site of development is not just BM, but also tisues referred to as primary and secondary lymphoid organs

In humans, the primary lymphoid organs are the thymus and bone marrow, the secondary organs include the spleen, Peyer’s patches of the GI tract, the Waldermyer ring of the tonsils and adenoids, the lymph nodes and modules scattered throughout the body

04/13/2346

LymphocytesLymphocytes

Lymphocytes circulate throughout the body in both PB and lymph which act as carrier streams to bring the lymphocytes to sites of activity

Lymphocytes migrate from thoracic duct through vessel endothelium to lymph nodes to blood stream and back.

Lymphocytes are categorized in a variety of ways and may be short-lived or long-lived cells

Lymphocytes may produce antibodies or lymphokines and have different surface charges, densities and antigen receptors.

04/13/2347

Lymphocytes - DevelopmentLymphocytes - Development

The PSC results in a stem cell for the lymphoid cell (CFU-L) as a result of hormonal stimuli

The CFU-L matures in several environments

Thymus and BM give rise to lymphocytes, foster differentiation and are indepentendent of antigenic stimulation

04/13/2348


Cells that develop under the influence of the thymus are called T cells and have specific receptors and responses.

B cells develop from the BM and have a different set of functions and receptors.

The end cell of the B lymphocyte maturation is the plasma cell

Once the environmental effects of the thymus and BM have been achieved, lymphocytes migrate to secondary lymphatic tissues such as the spleen and tonsils, which act as the main repositories for already differentiated lymphocytes.

04/13/2349


Cellular interactions for the presentation of antigen to the cells have a critical role in priming cells for proliferation and impact cell maturation, especially T cells. Once primed, the cells are now responsive to specific antigens.

Lymphocytes demonstrate lymphoblast, prolymphocyte and mature lymphocyte stages when stained with Romanowsky stains.

04/13/2350


Lymphocyte % in the PB varies, depending on age.

Children under the age of 4 have a higher proportion of lymphocytes in the PB than do adults

Lymphocytes are the second most common WBC of the PB making up 20-40% of WBCs.

20-35% of circulating lymphocytes are B cells

04/13/2351

Lymphocytes - MaturationLymphocytes - Maturation

Lymphoblast to prolymphocyteLymphoblast is small, 10-18 umRound to oval nucleusLoose chromatin with one or more active

nucleoliScanty cytoplasmProlymphocyte difficult to distinguish,

subtle changes, more clumped chromatin, lessening nucleolar priminence, change in thickness of the nuclear membrane

04/13/2352

Lymphocytes - MaturationLymphocytes - Maturation

Prolymphocyte to Lymphocyte Lymphocytes vary mostly by size1. Small – 9 um in diameter, non-dividing

or resting2. Medium – 11-14 um, non-dividing3. Large – 15 um, more generous cytoplasm

that is deep blue when stained Morphologic variants (table 11-2)

04/13/2353

Lymphocytes – Immunologic Lymphocytes – Immunologic DifferentiationDifferentiation Lymphocytes may be classified by

immunologic function B Cells1. Possess cytoplasmic IG concentrations of IgD

and IgM2. Some membrane receptors are apparent3. The fully committed B lymphocyte is the

plasma cell4. Demonstrate class I and class II human

leukocyte antigens (HLA-A, HLA-B, HLA-C and HLA-D, HLA-DR)

04/13/2354

Lymphocytes – Immunologic Lymphocytes – Immunologic DifferentiationDifferentiation T Cells1. The primitive T cell, CFU-L, travels to the

thymus2. Acquires a transferrin receptor that is specific

to proliferation3. Mature T cells lose all precursor markers and

have an active helper or suppressor function4. T cells are further differentiated through

presence or absence of HLA-D antigens5. T cells possess HLA-A, HLA-B and HLA-C

class I antigens

04/13/2355

Lymphocytes - ActivityLymphocytes - Activity

The main function of the lymphocyte is to regulate immune function

If foreign material is completely engulfed, degraded and disposed of by phagocytes, no immune response occurs

If digestion is incomplete, antigenic fragments are transported to lymph nodes

In the lymph node the antigen is fixed to the exterior surface and brought into the lysozymes of the macrophage

04/13/2356

Lymphocytes - ActivityLymphocytes - Activity

The antigen is processed and once that occurs, proliferation occurs

Development of clones of antigen-specific B lymphocytes and cytotoxic T cells begins

Activity that accompanies clonal expansion required for antigen removal can be seen in the morphology of cells called reactive lymphocytes

Hematology 425, Leukopoiesis

Documents

leukopoiesis leukopoiesis

leukopoiesis granulocytes

leukopoiesis wbcs

leukopoiesis lymphocytes

bone marrow granulocytes

pb granulocytes

granulocytes maturation

granulocytes bone marrow