Lecture 4: Hematopoiesis & Bone Marrow Evaluation

What is found in the hematopoietic microenvironment?

• stromal elements: endothelial cells, reticular cells, adipocytes, and ECM

• accessory cells: macrophages, T & B lymphocytes

• hematopoietic cells

What hematopoietic cells are found int he extravascular space?

• hematopoietic stem cells (HSC)

• hematopoietic progenitor cells (HPC)

• recognizable blood cell precursors

• mature leukocyte storage pools, especially neutrophils

What cells produce bone marrow stromal cells?

mesenchymal stem cells

What cells regulate the transendothelial movement of cells between blood and the marrow extravascular space?

endothelial cells

What fibroblastic-type cells provide structural support for the bone marrow?

reticular cells

What roles do endothelial cells and reticular cells share concerning bone marrow?

• synthesis of growth factors and ECM

• supports hematopoiesis through cell-cell contact

How do blood cells enter bone marrow?

enter the vascular sinus by passing through endothelial cells

What are HSCs generally positive for?

the CD34 surface antigen

How do HSCs replicate?

sustained self-replication slowly (once every 8-10 wks)

What do HSCs have the capacity to differentiate into?

all blood cell types and some tissue cell types (macrophages, DCs, osteoclasts, mast cells)

What common lymphoid progenitors (CLPs) are produced by HSCs?

B lymphocytes, T lymphocytes, NK cells

What common myeloid progenitors (CMP) are produced by HSCs?

non-lymphoid blood cells, macrophages, DCs, osteoclasts, mast cells

What common progenitor produced by HSCs is needed to form platelets and erythrocytes?

common MYELOID progenitor

Why do HPCs have limited self-replication?

they’re not sustained without replenishment from HSCs

How does HPC differentiation differ than that of HSC?

more restricted lineage potential

What are hematopoietic growth factors (HGFs)?

glycoprotein cytokines that promote the proliferation, maturation, and survival of hematopoietic cells

Where are HGFs produced?

locally in marrow (paracrine and autocrine) and/or by cells in peripheral tissues (endocrine)

What is an example of an endocrine HGF?

erythropoietin from kidney

What are the early-acting hematopoietic growth factors?

stem cell factor (SCF) and flt3 ligand (FL)

What are the intermediate-acting hematopoietic growth factors?

IL3 and GM-CSF

What are the late-acting hematopoietic growth factors?

G-CSF, M-CSF, EPO, TPO, and IL5

How does indirect stimulation cause HGF release?

during inflammation, TNFalpha and IL1 stimulate stromal and accessory cells to release various HGFs

What are erythroid islands?

microenvironment in bone marrow created by macrophages where erythroid precursors develop

What is the function of erythroid islands?

• phagocytize expelled nuclei of damaged or aged erythrocytes

• store iron released from phagocytized erythrocytes

What can be seen in the bone marrow sample? Note the central macrophage.

erythroid islands

At what maturational division does marrow release in dogs?

reticulocytes

At what maturational division does marrow release in horses?

erythrocytes

What is formed when metarubricytes expel their nuclei, usually while still bound to macrophages?

reticulocytes

How many reticulocytes does one rubriblast produce?

16 reticulocytes in about 4 days

What is the glycoprotein growth factor that promotes proliferation, differentiation, and survival erythroid progenitor cells and early erythroid precursors?

erythropoietin (EPO)

What is the main mechanism of action EPO?

inhibition of apoptosis

What are the sites of EPO synthesis?

• renal interstitial cells (primary site in adults

• extrarenal production: liver in mammalian fetus, bone marrow macrophages and erythroid cells

What effect does tissue hypoxia in the kidney have on EPO?

increased synthesis

What are the stages of erythropoiesis?

rubriblast → prorubricyte → basophilic rubricyte → polychromatophilic rubricyte → metarubricyte → polychromatophilic erythrocyte → erythrocyte

At what stage of erythropoiesis is a nucleoli present?

rubriblast

At what stage of erythropoiesis does division stop?

after polychromatophilic rubricyte

What are the stages of neutrophil production?

myeloblast → promyelo → myelocyte → metamyelocytes → band → neutrophil

At what point in neutrophil production does mitosis stop and maturation and storage begins?

between myelocyte and metamylocytes

What is the function of G-CSF?

• granulocyte progenitors to myeloblasts

• increased cell division, decrease marrow transit time

What cells produce various neutrophilic growth factors?

• fibroblasts and endothelial cells

• T lymphocyte and mononuclear phagocytes

What causes terminal maturation of eosinophils?

IL-5 from Th2 lymphocytes

Where do mast cells develop?

in tissues

What promotes development of mast cells?

stem cell factor

What late activating factor stimulates monocyte production/

M-CSF

What may monocytes become?

• macrophages or inflammatory dendritic cells in tissues

• osteoclasts in bone marrow

• dependent on amount of various cytokines present

What are osteoclasts?

multinucleated cells developed from monocyte fusion that degrade bone

Where are B-cells produced?

• marrow in most mammals

• Peyer’s patches in dogs, pigs, and ruminants

• bursa of fabricius in birds

Where are T-cells produced?

progenitors in marrow, but final development is in the thymus

Where are NK cells produced?

primarily in bone marrow and subsets develop in tissues

What is produced by thrombopoiesis?

megakaryocyte

True or false: non mammals do not have megakaryocytes.

true

What occurs in the megakaryocyte niche?

proplatelet processes (tube-like extensions) extend off of the megkaryocyte into the vascular sinuses and platelets form from the proplatelet process within the vasculature; platelets sheared off when matured

How does thrombopoietin (TPO) stimulate thrombopoiesis?

• promotes differentiation of progenitor cells into megakaryocytes

• stimulates increased endomitosis, resulting in increased ploidy (number of sets of chromosomes) and size

What is the major site of production of thrombopoietin?

endothelial cells of liver

What stimulates TPO synthesis by the liver during inflammation?

inflammatory cytokine IL-6

Why do recombinant growth factors only work for a short period of time?

antibodies are made against these glycoproteins, which limits their effectiveness

What are some reasons for bone marrow examinations?

• proliferative abnormalities with unexplained left shifts, nucleated erythroid cells, or blast cells in blood

• evaluate iron stores (except in cats)

• unexplained hyper proteinemia in blood

What are some bone marrow biopsy sites for small animals?

• dorsal iliac crest

• wing of ilium

• proximal humerus

• trochanteric fossa of the femur

What is contained in a bone marrow aspiration syringe?

EDTA in a sterile saline solution

When would a bone marrow core biopsy be done?

• to get better evaluation of overall cellularity after unsuccessful aspirate attempts

• identifcation of focal lesions that may occur with metastatic neoplasia, necrosis, or granulomatous inflammation

What is used to evaluate the number, morphology, and stages of megakaryocyte development?

sytematic bone marrow examination

What is calculated in a systematic bone marrow examination?

• the M:E (granulocytic:erythrocytic) ratio

• amount of hemosiderin is assessed too

What is the arrow pointing to in this bone marrow sample?

megakaryocytes

promegakaryocyte

basophilic megakaryocyte

mature megakaryocyte

polychromatophilic rubricyte

rubriblast

myeloblast

promyelocyte

myelocytes

band eosinophil

basophil

What knowledge is essential to interpret the M:E ratio?

Hct and total neutrophil count in blood

If the absolute blood neutrophil count is normal, what do changes in the M:E ratio reflect>

changes in the erythroid series

If Hct is normal, what do changes in M:E generally reflect?

changes in the neutrophilic series

When is the M:E ratio of little value?

if the marrow is markedly hypoplastic or filled with neoplastic cell

What are these two monocyte precursor cells?

left: monoblast

right: promonocyte

plasma cells

True or false: stainable iron is present in normal cat bone marrow aspirates.

false