Lecture/Lab: Hematopoiesis & Bone Marrow Flashcards
Sites of Hematopoiesis
Yolk Sac, Liver, Bone Marrow, Red Marrow
Yolk Sac Hematopoiesis
extraembryonic site
hematopoietic islands form here in the 3rd-4th week of gestation, peaking in 2nd month
Liver (and Spleen) Hematopoiesis
hematopoiesis begins here in the 5th week, peaks at 5-6 months gestation
Bone Marrow Hematopoiesis
hematopoiesis begins here in the
5th month of gestation, continues to adulthood
Red Marrow Hematopoiesis
limited to specific locations after
puberty
Location of RM: heads of long bones, rib cage, vertebral columns, pelvis, sternum
After puberty, red marrow typically is confined to the metaphyseal regions of long bones and to the axial skeleton. The crest of the iliac bone (the prominence one feels at one’s hips) is the preferred site for marrow biopsy as it is the part of the axial skeleton furthest away from CNS
Yellow Marrow vs. Red Marrow
Yellow- Higher proportion of adipose tissue
Red- Higher proportion of Hematopoietic cells
Macrophage Sequestration (Storage)
- stored in spleen (as monocytes)
- rapid deployment during tissue repair (ie myocardial infarction)
Neutrophil Sequestration (Storage)
- “Marginated pool” PMNs (neutrophils) are adherent to endothelial cells
- Postcapillary venules of lung is largest site of marginated (adhered) pool
- normally 1/2 of PMNs are marginated (explains why CBC doesn’t tell the whole story)
- released in response to stress,
epinephrine
Lymphocyte Sequestration (Storage)
stored in secondary lymphatic organs (MALT, BALT)
Vasculogenesis
-The creation of a vessel out of CT
-MET event (mesenchymal to epithelial transition)
-Embryonic event, doesn’t happen in adults
Extraembryonic Hematopoiesis
Early blood and vessels come from the same source in extraembryonic (lateral sphlancnic) mesoderm
Blood island
collection of 1st progenitor cells of blood
Angiogenesis
-The sprouting of vessels from existing vessels
-Happens in adults
-Occurs in response to lack of oxygen
Tip cells
special endothelial cells that direct the formation of new vessels (via angiogenesis) by pulling stalk cells into vascular lumens
Hematopoietic tissue composition
The composition of hematopoietic tissue changes based on site, particularly for monocytes, neutrophils and dendritic cells, which are first found in bone marrow
This is expected because the needs of the embryo, fetus, and adult are different
Progenitors of the “organ-colonizing” cells are specific to liver
Composition of the vascular compartment of bone marrow
vascular sinus
(large, sinusoidal capillaries)
Composition of the hematopoietic compartment of bone marrow
stroma (supporting tissue):
- adipose tissue
- fibroblasts
- connective tissue
- endothelium
parenchyma (“functional” tissue):
- developing blood cells (hematopoietic islands)
Origin of each type of stromal cell
Derived from MSC
- Osteoblasts (secrete collagen & fibronectin for new bone)
- Adipocytes (store fat)
- Fibroblasts (secrete ECM)
- Endothelial cells (vascular lining)
Derived from HSC
- Osteoclasts (secrete protons, etc., to break down bone)
- Macrophages (phagocytic)
Stem cell
From Video:
A cell that can both renew itself (divide and create a daughter cell that is also a stem cell) and is totipotent (has ability to generate all of the cell lines that are found within that system)
ex: a cell that can create all of the cells of blood
Progenitor cell
From Video:
A cell that can renew itself, but cannot produce the whole lineage (multipotent)
ex: a cell that can produce lymphocytes, but none of the other blood cells
Precursor cell
From Video:
Cannot renew itself. If it divides, differentiation will go along with it. The daughter cells must be more differentiated than the parent cell. Restricted to one particular lineage of adult cells.
What two classes of progenitor cells are produced from hematopoietic stem cells? (classical view)
Common Myeloid Progenitor (CMP)
- erythrocytes
- platelets
- macrophages
- granulocytes
Common Lymphoid Progenitor (CLP)
- lymphocytes
What cells are produced from each class of progenitor cells? (classical view)
CMP (common lymphoid progenitor) which gives rise to:
– CFU- Eo (eosinophils)
– CFU- B (basophils)
– CFU -GM –> CFU-G (neutrophils) & CFU-M (monocytes)
– CFU-Megakaryocyte
– BFU-E (burst-forming unit - erythrocyte) –>CFU-E (erythrocytes)
CLP (common lymphoid progenitor) which gives rise to:
– CFU- LyT
– CFU- LyB
What are the precursor cells in blood and what cells result from them?
The first precursor cell in each lineage is a “blast”.
– Proerythroblasts ultimately form erythrocytes.
– Myeloblasts ultimately form granulocytes.
What are CD markers and why are they important?
Because stem cells and progenitor cells are not morphologically distinguishable, they are recognized (and defined) in laboratories by using combinations of CD markers, made from monoclonal antibodies for surface proteins. CD markers such as CD-4 and CD-8 are famously used to distinguish different subtypes of T-cells. (from lecture notes)
presence (+) or absence (-) or even
the level of expression (lo, hi) of a
CD molecule can be used to define
a developmental stage.
CD expression can be dependent
on lineage and/or on stage of
differentiation.
CD4+ (Helper T cell)
CD8+ (Cytotoxic T cell)
CD3+ part of developing T cells
What are the erythrocyte precursor cells?
Proerythroblast, Basophilic erythroblast, Polychromatic erythroblast, Orthochromatophilic erythroblast, and reticulocyte
What is the main way to distinguish between erythrocyte precursor cells?
precursor cells are chiefly distinguished by their cytoplasmic coloration
Less important characteristics are the size of the cell (goes from large to small), the size of the nucleus (big to small), the chromatin (euchromatic to heterochromatic)
Basophilic erythroblast
Contains all ribosomes, which make the cytoplasm basophilic
We need a lot of ribosomes to make proteins (hemoglobin)
Fine chromatin clumps in nucleus, basophilic cytoplasm
Large (usually slightly smaller) cell with basophilic cytoplasm but lacking nucleoli
Polychromatic erythroblast
Begins to produce hemoglobin which is eosinophilic
mix of ribosomes & hemoglobin
Coarsechromatin clumps in nucleus, grey-blue cytoplasm, last stage for mitosis
average-sized cell with a cytoplasm intermediate in color between basophilic and eosinophilic extremes
Orthochromatophilic erythroblast
Means “identical color”
Same color as erythrocyte
all hemoglobin
average-sized cell with a cytoplasm equivalent in color to that of the reticulocytes
Reticulocyte
No nucleus, but does have some remnant organelles (not visible)
On marrow smear, assume all RBCs are reticulocytes
small, spherical eosinophilic cell lacking a nucleus. Remnants of intracellular organelles can be visualized with special stains
Nucleus gone, fine strands in cytoplasm, frequency in peripheral blood depends on hematopoietic activity
Shape is intermediate between spheroid and biconcave RNA and intracellular organelles still retained
Not easily visualized in WrightGiesma stains – methylene blue is typically used for reticulocyte counts
Very important part of a clinical
blood workup. They will count reticulocytes which should be around 2%
Erythropoiesis
Erythropoiesis is the process by which red blood cells (RBCs) are produced in the bone marrow
Erythropoiesis is regulated by substances normally present in marrow stroma:
– G-CSF (granulocyte colony stimulating factor),
– SCF (stem cell factor)
– IL3
– The cytokine Erythropoietin (EPO) (made in kidney corticointerstitial cells), which is itself regulated by:
* Oxygen levels
* Renin-Angiotensin system (sensing blood pressure)
* Other hormones, for example insulin
Erythrocyte progenitor cells
CMP – generates all non-lymphoid lineages.
BFU-E – progenitor cell
- requires several cytokines for survival
- high proliferative potential
- 14 days to RBCs in culture
- motile – can be present in peripheral blood
CFU-E – progenitor cell
- highly dependent on EPO
- 7 days to RBCs in culture
- non-motile, found in marrow only
Proerythroblast
Nucleoli present in nucleus,
no granules in cytoplasm
Large cell with nucleoli and cytoplasm that can range from neutral to basophilic. In common preparations, it is not distinguishable from the myeloblast
What does increased reticulocyte count mean?
internal bleed or chronic anemia because marrow is pumping lots of reticulocytes into blood
How to reticulocytes and granulocytes leave the marrow?
Reticulocytes leave the marrow by piercing through endothelial cells
Mitochondria and polysomes still finishing the production of hemoglobin
Biconcave erythrocyte shape is formed within the circulation
Reticulocytes leave the marrow, and are normally present at < 1.5% in peripheral blood.
Granulocytes leave the marrow as either band cells or mature forms. Band cells are normally present at about 3% in peripheral blood.
What are the 3 types of granules that neutrophils have?
primary (azurophilic): lysosomes
secondary (specific, i.e. neutrophilic,
eosinophilic, basophilic)
tertiary: chemotactic
How to distinguish blast cells? (proerythroblast and myeloblast)
Large cell, large nucleus, prominent nucleolus (large white spaces)
What is the order of the neutrophil and eosinophil lineage?
Myeloblast –> Promyelocyte –> Early neutrophilic/eosinophilic myelocyte –> Late neutrophilic/eosinophilic myelocyte –> Neutrophilic/Eosinophilic metamyelocyte –> Band cell (only for neutrophils) –> Mature neutrophil/eosinophil
What type of granule is present in each stage in the granulocyte lineage?
Myeloblast: no granules present
Promyelocyte: primary (azurophilic) granules
Early neutrophilic/eosinophilic myelocyte: secondary (specific) granules
Early neutrophilic/eosinophilic myelocyte
round nucleus
specific granules begin to accumulate intracellularly.
few specific granules
Late neutrophilic/eosinophilic myelocyte
kidney shape nucleus
many specific granules
Neutrophilic/Eosinophilic metamyelocyte
V-shape nucleus
V-shaped nucleus, i.e. containing an acute angle indentation. Basophilic metamyelocytes are not normally distinguished in LM because the basophilic granules (unlike eosinophilic granules) obscure the outline of the nucleus.
Band cell
C-shaped nucleus, without chromatin
bridges
immature form of neutrophil normally present in peripheral blood in small quantities, but over 2% can indicate pathology
C-shaped nucleus (usually no acute indentations), but be careful to consider the orientation of the cell on the slide. Normally present at about 2% in peripheral blood.
Monocyte development
Progenitors: CFU_GM, CFU-M
Precursors: monoblast, promonocyte
In blood: circulating monocyte
In tissue: macrophages, dendritic cells, osteoclasts
Lymphocyte development
Progenitors: CFU-Ly, CFU-LyB, CFU-LyT
Precursors: lymphoblast, prolymphocyte
In blood: circulating lymphocytes
In tissue: Ly-B (B lymphocyte), Ly-T (T T lymphocyte), NK-cells (natural killer cells), interstitial plasma cells
Megakaryocytes
- Reside exclusively in bone marrow, are HUGE cells (100 μm in diameter)
- Release pre-platelets into sinusoid/sinus capillary, which mature into platelets in circulation
- Polyploid cells (one nucleus but many copies of DNA) as a result of endomitotic division in megakaryoblasts
- Megakaryocytes with higher chromosome content produce more platelets than megakaryocytes with low chromosome content.
- Platelet demarcation channels form in cytoplasm
- 8000 platelets produced per
megakaryocyte - Platelets (pre-platelets) are pinched off within the lumen
Life cycle of a platelet
platelets are pinched off from megakaryocytes in bone marrow & released into circulation
(megakaryocytes are capable of altering the rate of platelet production over an entire order of magnitude)
activated in response to clotting factors
typically activated outside the circulation, or by exposure to basal
surface of endothelia
normally last about 10 days *
“stored” in circulation
Primary lymphoid organs
Bone Marrow and Thymus
Responsible for generating and maturing lymphocytes
Anemia
disorders of RBCs
Reduction in the oxygen carrying capacity of the blood
Causes include:
– Blood loss via trauma or genetic defects
– Kidney disease (kidney and liver make erythropoietin)
– Bone marrow cancer
Too few RBCs
- Too few produced
- Too many lost or destroyed
Too many RBCs (polycythemia)
- Right number of RBCs, but:
Too small
Too large
Wrong shape (ex: sickle-cell anemia)
Leukemia
disorders of WBCs
Basic classification of leukemia
First letter:
– A for Acute = few mature circulating WBCs
– C for Chronic = abnormal function in circulating WBCs
Second letter (which progenitor):
– M for Myelogenous = affecting myeloid cells
– L for Lymphocytic = affecting lymphocyte cells
Third letter:
– L for Leukemia = abnormal proliferation of leukocytes, sometimes called ‘cancer of the blood’
Acute Leukemias
Very few mature leukocytes (WBC’s) in blood. Blood may contain cells normally found in marrow.
Marrow is full of rapidly dividing immature cells that do not differentiate.
Anemia and bleeding are common as normal cells in marrow are affected by crowding.
Treatments include drugs, radiation, bone marrow transplants and autologous stem cell transplants.
promyelocyte
smaller cell with nucleoli and azurophilic granules appearing, often near the Golgi.
mature neutrophil, eosinophil, basophil
segmented nucleus. Granulocytes usually leave marrow in their mature form, though in case of demand, late precursors may also leave.
Platelet demarcation channels
cytoplasmic structures which precede the formation of the proplatelet strings, are recognizable EM features of megakaryocytes
