Intro to Hematopoiesis

methemoglobin reductase pathway

- (this is the branch point immediately below the HMP shunt in the above diagram) also helps protect RBCs against oxidative injury. - Methemoglobin (Hgb- Fe3+) is hemoglobin that contains non- functional oxidized ferric iron (Fe3+) rather than the functional form of ferrous iron (Fe2+). Methemoglobin is considered non-functional because it cannot bind oxygen; however, this change is reversible. NADH produced in the glycolytic pathway is a cofactor for the enzyme methemoglobin reductase, which reverses the oxidation of iron and returns it to the normal reduced (Fe2+) state. - Note in the diagram above MB and LMB refer to the reversal of methemoglobin that occurs when new- methylene blue is used therapeutically.

what are oxygen levels in the bone marrow?

- 7-31 mmHg (1-4%) - The relative hypoxia in the bone marrow helps maintain the high proliferation capacity of the cells in the marrow

how does hypoxia impact iron transport?

- At the level of the kindey, once the EPO starts to promote expansion of the erythroblast population, then erythroferrone is produced by RBC precursors, and this will suppress hepcidin, allowing a release on the iron hold. - Hypoxia can also impact iron transport independently. The transporters on the luminal side of the enterocytes that take up iron (DMT1 etc) as well as ferroportin on the basal side, are increased by HIF - 2α. If an animal is hypoxic, the body "prepares" to make more RBC by upregulating pathways to acquire iron.

what is the result of RBCs carrying so much oxygen?

- Because RBCs carry so much oxygen, they are prone to oxidative injury. - Normally, as oxygen binds to and is released from the iron in Hgb, superoxide radicals can be formed in small amounts. - RBCs have antioxidant mechanisms to deal with this as long as it is not excessive (more on this to follow). However, exposure to certain exogenous oxidative compounds can overwhelm the RBC antioxidant capability and lead to oxidative damage.

Heinz bodies

- G6PD deficiency and thalassemia - Heinz body formation is an etiology of hemolytic anemia. - Irreversible oxidation of sulfhydryl groups within globin of Hgb - causes denaturation and precipitation of Hgb into visible pale or protruding areas within RBC

what are the two key mediators that regulate ferroportin

- Hepcidin: A protein made predominantly in the liver (low levels are made in macrophages and adipocytes). Hepcidin is produced under the stimulus of inflammatory cytokines and iron excess. When hepcidin is produced it acts to decrease ferroportin, which traps iron in the macrophages. This is a leading mechanism that contributes to the anemia (decrease in the number of functional erythrocytes) that is often seen in the context of inflammatory disease. Ferroportin is also the transport protein used by the intestinal epithelial cells to absorb iron into the blood. Hepcidin also serves to decrease intestinal absorption by decreasing ferroportin (via promoting its destruction) on intestinal cells. - Erythroferrone (EFN): suppresses hepcidin production. EFN basically releases the hold on iron by decreasing hepcidin. EFN will therefore increase intestinal absorption and release of iron from macrophages. EPO drives the production of erythroferrone in the early erythroid precursors (thought to be mostly erythroblasts) in both the bone marrow and spleen.

what is unique about the horse (anemia)?

- Horses very seldom release reticulocytes into circulation during diseases that cause increased RBC loss or destruction (although they do release mature RBCs that are slightly larger than normal), so you cannot rely on reticulocyte counts to define a regenerative anemia in a horse. - Instead, we have to look for a higher MCV and higher RDW due to release of larger RBCs into circulation.

why is there a decrease in hematopoiesis in the obese and elderly?

- In the average adult, 10% of the total body fat resides within the marrow, and accounts for approximately 70% of the bone marrow's volume. Marrow adipose tissue, unlike fat beds in soft tissues, is confined by the margins of the skeleton. Therefore, if the adipose tissue expands, there is less room for hematopoiesis.

ferroportin

- In the spleen and liver, macrophages ingest old RBC and break them down. The iron is released from these macrophages via a transmembrane transport protein called ferroportin .

how do macrophages aquire and release iron?

- In the spleen and liver, macrophages ingest old RBC and break them down. The iron is released from these macrophages via a transmembrane transport protein called ferroportin . - As the iron exits the macrophage, it binds to a carrier protein in the plasma called transferrin. -The iron-transferrin complex travels via the blood stream to the bone marrow and enters a macrophage via the transferrin receptor. Iron and free hemoglobin (where might free hemoglobin be coming from?) can also enter a nurse macrophage via the DMT1 transporters and various scavenger receptors. - The macrophages in the bone marrow have an arsenal of receptors to pull in iron/hemoglobin/RBC. W ithin the macrophages, the iron can be used by the macrophages for selfish purposes, stored (as ferritin), or released into developing RBC precursors. The release of iron from the macrophages, into the developing RBC is thought to occur via the ferroportin receptor (there are other receptors under investigation). Once inside the erythroid progenitor cell, the iron traffics to the mitochondria, where it is then incorporated in to hemoglobin (the protein components of which are synthesized on ribosomes) and other iro n - containing proteins. The function of hemoglobin is covered later in the notes.

Do RBC's have mitochondria? If not how do they get their needed ATP molecules?

- Mature RBC lack mitochondria (the site of aerobic phosphorylation), hence they depend on anaerobic glycolysis to produce ATP. - There are three main branch points off the glycolytic pathway that produce various other compounds that are necessary within the RBCs: the hexose monophosphate shunt (also called the pentose phosphate pethway), the methemoglobin reductase pathway, and the Luebering-Rapaport pathway (which is commonly referred to as the 2,3- DPG shunt).

Extravascular hemolysis

- RBC does not lyse or break apart until after it has been taken up by macrophages

what are important oxidative compounds? what are the result of ingestion/contact with these compounds?

- Some of the more clinically important oxidative compounds include onions, wilted red maple leaves (horses), acetaminophen, and various other drugs/toxic plants. - Endogenous oxidants are produced in disease states include inflammation, diabetes and neoplasia. Lipids in the cell membrane and thiol (SH, sulfhydryl groups) are especially sensitive to oxidation. - If membrane lipids/proteins are oxidatively-injured they may stick together and lead to the formation of eccentrocytes. Similarly, if hemoglobin is oxidized and precipitates, this can lead to the formation of Heinz bodies.

what parts of the RBC are degraded and recycled with uptake by macrophages?

- The amino acids from the globin portion of Hgb are recycled and can be used again. - The heme portion is broken down into iron and porphyrin. - The iron is recycled and can be used again in the bone marrow for production of new RBCs. - The porphyrin is metabolized to bilirubin, which is conjugated in the liver and excreted in the bile .

why is ATP required by the RBC?

- The generation of ATP and metabolic intermediates allows the RBC to maintain membrane integrity, cell size, and redox status. - ATP is required to maintain cell shape and deform ability, since it is needed to regulate water and electrolyte content (which affects size and shape of the cell) and to maintain the cytoskeleton (which is needed for proper deformability). As a result, RBCs can lyse when depleted of ATP.

explain how red blood cells carry out their function

- The primary functional role of the RBC is to transport oxygen from the lungs to the tissues, and carbon dioxide from tissues to the lungs for expulsion. - This functional role is served by hemoglobin. Each Hgb molecule is composed of 4 iron-containing heme units and 4 polypeptide globin units. - The heme unit is a porphyrin ring and is the portion of the hemoglobin molecule that binds iron. The globin unit consists of polypeptides and functions in part to prevent the iron- containing heme molecules from coming in close contact with each other, which would interfere with function. O2 binds reversibly to the iron within the heme molecule, and is released in tissues (promoted by multiple factors including 2,3- DPG). CO2 is transported from the tissues back to the lungs in 3 ways: some (about 20- 30%) is transported reversibly bound to amine groups in hemoglobin (not to iron), a small amount (about 7% ) is transported as dissolved CO 2 in the blood, and most (about 70% ) is transported inside the red cells as carbonic acid produced from CO2 and water by carbonic anhydrase.

role of hemoglobin in a RBC

- The primary functional role of the RBC is to transport oxygen from the lungs to the tissues, and carbon dioxide from tissues to the lungs for expulsion. - This functional role is served by hemoglobin. Each Hgb molecule is composed of 4 iron-containing heme units and 4 polypeptide globin units. - The heme unit is a porphyrin ring and is the portion of the hemoglobin molecule that binds iron. The globin unit consists of polypeptides and functions in part to prevent the iron- containing heme molecules from coming in close contact with each other, which would interfere with function. O2 binds reversibly to the iron within the heme molecule, and is released in tissues (promoted by multiple factors including 2,3- DPG). CO2 is transported from the tissues back to the lungs in 3 ways: some (about 20- 30%) is transported reversibly bound to amine groups in hemoglobin (not to iron), a small amount (about 7% ) is transported as dissolved CO 2 in the blood, and most (about 70% ) is transported inside the red cells as carbonic acid produced from CO2 and water by carbonic anhydrase. - Hemoglobin also provides viscosity to the RBC cytoplasm. This is crucial in maintenance of cell shape and membrane stability. In state of iron deficiency, the decrease in cytoplasmic hemoglobin is thought to contribute to a decrease in cytoplasmic viscosity, and this is followed by membrane instability (hence we can often see cellular fragmentation in severe iron deficiency).

fatty acid oxidation

- The signals that drive asymmetric division of stem cells, where one cell differentiates into a daughter cell and the other retains stem cell properties, are not fully characterized. However, fatty-acid oxidation has been shown to be a key regulatory switch in this pathway and serves largely to retain the stem cell nature of the HSC. - The switch from fatty acid oxidation (FAO) to oxidative phosphorylation (OXPHOS) is concurrent with differentiation of the HSC and a decreased potential for self-renewal.

what is a benefit of glycolysis over aerobic respiration?

- While glycolysis yield s far less ATP than aerobic respiration, it generates more NADPH which serves as a building block in the synthesis of macromolecules such as amino acids and nucleotides that are required for cell division. - The preference HSC glycolytic pathways reduces the production of reactive oxygen species (ROS). Lower levels of ROS helps protect the HSC from injury, especially mitochondrial damage.

Metarubricyte

- a changed red cell - rubriblasts through metarubricytes are nucleated RBCs (nRBCs). In mammals, metarubricytes then extrude their nucleus (through a complex process that involves parts of apoptotic pathways but does not result in cell death), so reticulocytes and mature erythrocytes lack a nucleus in mammals. In non-mammalian species, all erythroid stages are nucleated.

anemia

- a decrease in RBC mass - reflected by a low PCV/HCT and hemoglobin - result of either increased loss/destruction (hemorrhage or hemolysis) of RBC or decreased producetion of erythroid cells in the bone marrow

Hypoxia-inducible factor (HIF)

- a family of transcription regulators that coordinate the expression of many genes in response to oxygen deprivation - Hypoxic activates (or sta b ilize s), the hypoxia- inducible factors (HIF) 1α and 2α transcription factors. HIF-1α is expressed ubiquitously, whereas HIF-2α is m ore restricted but i s expressed in all hematopoietic cells. In concert, the HIFs in the bone marrow function to keep the cells in a glycolytic state by limiting production of the proteins involved in the TCA cycle. W hile glycolysis yie ld s far less ATP than aerobic respiration, it generates more NADPH which serves as a building block in the synthesis of macromolecules such as amino acids and nucleotides that are required for cell division. Thus, the metabolism of the HSC is shifted towards m aking more cells rather than individual metabolic activity.

eythroblastic islands

- a nurse macrophage provides the iron and other secreted factors needed by the developing cells and will phagocytose the extruded nucleus (called a pyrenocyte). - once a reticulocyte is formed, it stays in the bone marrow for approximately 24 hours (in people, we do not know precisely how long this stage takes in our common domestic species) and then the cell is released into circulation. The plasma membrane composition of the RBC changes to facilitate this release, largely by decreasing the expression of, and cleaving off, adhesion molecules. Reticulocytes released into circulation can further mature to a RBC by removal of internal organelles (largely the ribosom es that were essential for hemoglobin synthesis). This is accomplished largely by splenic macrophages. If the condensation of the nucleus is not temporally linked with hemoglobinization of the cytoplasm, this is recognized as a dysplastic change and often referred to as asynchronous maturation.

intravascular hemolysis

- aged or damaged RBCs rupture while still in circulation - can occur when RBC are ATP depleted

what happens to the structure of the RBC with age/damage?

- become denser and less deformable - accumulation of oxidant injury to hemoglobin, which promotes surface-bound immunoglobulins - invert their lipid membrane amd expose phosphatidyl serire on their membrane (eryptosis), leading to uptake of RBC by macrophage - decreased ability to produce ATP, leading to decreased membrane deformability (signals splenic macrophages to remove RBCs)

what shape is the red blood cell in mammals? why?

- biconcave disk - increases the deformability of the cell, allowing it to get through very small capillaries and maximizes the surface area to volume ratio (increasing the obility for oxygen and carbon dioxide to diffuse in and out)

what clears free hemoglobin from circulation?

- binding to carrier proteins such as haptoglobin - the hemoglobin:haptoglobin complexes are taken up via scavenger recpetors into the macrophages

regenerative anemia

- caused by hemorrhage or hemolysis - characterized by an increase in polychromatophils - occurs because the marrow can respond to yhe demand

nonregenerative anemia

- causes of decreased production of erythoid cells can be due to a problem within the bone marrow (eg the stroma is damaged, there is cancer in the marrow, there is not enough iron to support erythropoiesis), or a disease originating outside of the marrow but having a negative impact on the ability of the bone marrow to produce erythrocytes (inflammation and renal disease fall into this category). - Polychromatophils are not increased in circulation.

Erythropoietin (EPO)

- class 1 cytokine hormone produced in the kidneys, liver, spleen, brain and testes; stimulates red blood cell formation - travels in the blood to the bone marrow where is binds to EPO receptor that is highly expressed on early stage RBC precursors. Ligation of the EPO receptor stimulate proliferation and differentiation of the erythroid precursor cells, and also inhibits apoptosis of erythroid progenitors. The net effect is increased production of RBC. In most animals, this wave of production results in an increase in polychromatophils in circulation. - transcription factor hypoxia inducible factor-1 (HIF- 1α) binds to the promoter region of the EPO gene and drives expression of EPO, largely in peritubular fibroblasts close to the cortico- medullary junction (recent studies suggest intercalated cells in cortical nephrons may also produce EPO). When oxygen is present, HIF is enzymatically destroyed, and EPO is not (or is minimally) produced. This is one of nature 's most exquisite feedback loops. When there is hypoxia these fibroblasts can expand in numbers and the cells that are closer to the renal cortex begin to make EPO mRNA. If the hypoxia is chronic, the fibroblasts may become more myofibroblastic, and reduce their production of EPO .

how do cells in the kidney recognize hypoxia?

- decrease in RBC mass - reduced hemoglobin - dysfunctional hemoglobin - regional tissue damage that impacts blood delivery

pancytopenia

- deficiency of all types of blood cells - indicates primary bone marrow disease

pyrenocyte

- enveloped extruded nucleus - engulfed by bone marrow macrophages

extramedullary hematopoiesis

- hematopoiesis outside bone marrow (spleen & liver) - may result in mass forming lesions anywhere in the body

hexose monophosphase shunt

- important because it helps protect RBCs against oxidative injury. - the enzyme G6PD produces NADPH from glucose 6 - phosphate in the glycolytic pathway, which is utilized to help maintain glutathione (GSH) in a reduced state. - Reduced glutathione is an important antioxidant peptide, and can protect against oxidation of iron, Hgb sulfhydryl groups, or cell membrane proteins. This pathway is incredibly important in RBC as they lack the organelles other cells use to assist them in dealing with oxidative stress (nucleated cells can alter gene transcription and increase synthesis of proteins that are involved in anti-oxidant and protective pathways, such as the heat shock proteins).

regenerative anemia on CBC

- increased absolute reticulocyte count - increased MCV (not always noted) - increased RDW (not always noted) - decreased in MCHC (not always noted)

what is the most common cause of nonregenerative anemia?

- inflammation - Inflammatory cytokines skew the differentiation of HSC in the marrow towards myeloid lineages. Inflammatory cytokines induce hepatic synthesis of hepcidin, which as previously described results in iron sequestration a nd a decrease in gastrointestinal absorption of iron. TNF- α decreases EPO expression in the kidney, and also suppresses HIF- 1α signaling in the bone marrow, thus further blunting erythropoiesis in the marrow. Cytokines have been shown to interfere with the progressive development of erythroid cells in culture. For example, IFN - g amma has been shown to cause a down- regulation of the EPO receptor.

eccentrocytes

- irreversible oxidation and cross-linking of cell membrane proteins - causes oxidized portions of the membrane to stick together and form a cler area with the remaining hemoglobin pushed off to one side

mesenchymal stromal cells (MSCs)

- make up the marrow niche or the microenvironment - the MSCs include osteoblasts, chondrocytes, macrophages, adipocytes, endothelial cells and pericytes - Secreted cytokines (such as erythropoietin, which drives RBC development) act in concert with stromal cells and growth factors to drive differentiation of HSCs.

red blood cell structure

- must traverse bloodsteam at high speeds and maintain the perfect degree of fluiditiy to allow them to move through the vessel without breaking, and concurrently allow for efficient gas exchange across the cell membrane - the cytoskeleton of the RBC has a high tensile strength, but it also highly deformable. The mechanical properties and shape of the RBC allow the RBC to get through very small capillaries without being damaged, because the diameter of the RBC is larger than the diameter of the smallest capillaries it must traverse.

stress/emergency hematopoiesis

- pathological events trigger a different pattern of hematopoiesis, usually extramedullary production - Inflammatory cytokines trigger the macrophages in the red pulp of the spleen to secrete C-C chemokine ligand 2 (CCL- 2) and this recruits monocytes to the spleen. Once arrived, these blood-derived monocytes form niches that develop into erythroblastic islands and support erythropoiesis. TNF-α, rather than suppressing HIF-1α as it does in morrow, stabilizes the protein, thus enhancing EMH at this site. In the spleen, glucorticoids synergize with HIF- 1α to further drive proliferation of erythroid progenitors. If there is increased turn-over of RBC in the spleen, this results in an additional heme-dependent increase in the transcription factors that trigger stress erythropoiesis. - In the case of a sin g le , short term inflammatory event, the stress response from the spleen compensates for the suppression of erythropoiesis in the marrow. This allows the marrow to be myeloid dominant. Thus, there is an acute inflammatory response evident in the CBC, but not necessarily an anemia. If the inflammation continues, the suppression is likely to become the dominant pattern, and a nonregenerative anemia may become evident.

Eryptosis

- premature death of damaged erythrocytes - invert their lipid membrane amd expose phosphatidyl serire on their membrane (eryptosis), leading to uptake of RBC by macrophage

luebering-rapaport pathway

- produces 2,3- DPG from an intermediate within the glycolytic pathway. 2,3-DPG is an organic phosphate compound that functions to promote release of oxygen from hemoglobin into tissue. It binds with greater affinity to partially deoxygenated hemoglobin and promotes release of the remaining oxygen molecules bound to the hemoglobin. When 2,3- DPG is bound to Hgb, the dissociation curve of Hgb is shifted to the right. A decrease in pH (acidosis in the tissue, which occurs with increased production of lactate) and an increase in temperature have the same effect on the Hgb saturation curve (see insert).

thrombopoiesis

- production of platelets - Platelets are formed as small anucleate cytoplasmic fragments of their bone marrow precursor cell, the megakaryocyte. The developing immature megakaryocyte undergoes a switch from mitotic division to endomitosis, which is basically cell division with incomplete cytokinesis. Thus, the nucleus becomes polyploid and cells with end up with high DNA copy numbers (64N in people and 256N in mice) After multiple round of endomitosis, a proplatelet forms and this extends through the vascular sinus. Then, the cytoplasm fragments break off of the megakaryocytes in the forming the anucleated platelets. In addition to producing platelets, megakaryocytes secrete factors that help maintain HSC quiescence in the niche (these include TGF- B and CXCL- 4). - The main cytokine that leads to increased numbers of megakaryocytes in the bone marrow is thrombopoietin (TPO). TPO is a glycoprotein produced by the predominantly by the liver with small a mounts arising from the kidney. TPO binds predominantly to the cMPL receptor on megakaryocytes. Platelets can bind TPO and prevent it from going to the bone marro w. W hen platelets are consumed or lost, there is more free TPO in circulation and hence a drive for megakaryocytic hyperplasia. Thus, circulating TPO levels are inversely related to platelet mass. People with cirrhotic liver failure often have decreased TP O. However, studies do not support a relationship between acute liver injury or inflammation and TPO.

what marks the maturation of erythroid cells?

- progressive condensation, and ultimately extrusion of, the nucleus - The final stages of maturation are when the cells synthesis the hemoglobin that will allow them to transport oxygen in the body. - once an RBC becomes a mature erythrocyte, it can no longer produce Hgb

how do you identify a polychromatic reticulocyte?

- purple hue due to retained ribosomes and hemoglobin concurrently imparting a blue and red tone to the cytoplasm

methemoglobin

- reversible oxidation of Fe2+ to Fe3+ within Hgb - cannot bind oxygen, so causes mucous membranes and blood to appear brown

what is the result of severe anemia and hypoxia in the kidney?

- there is an increased drive for EPO secretion. - Once the EPO starts to promote expansion of the erythroblast population, then erythroferrone is produced by RBC precursors, and this will suppress hepcidin, allowing a release on the iron hold. - Hypoxia can also impact iron transport independently. The transporters on the luminal side of the enterocytes that take up iron (DMT1 etc) as well as ferroportin on the basal side, are increased by HIF - 2α. If an animal is hypoxic, the body "prepares" to make more RBC by upregulating pathways to acquire iron.

how long is the RBC lifespan in domestic species?

2-4 months (shorter in small mammals and longer in reptiles)

structure of hemoglobin

4 iron-containing heme units and 4 polypeptide globin units

what are oxygen levels in the peripheral blood?

75-100 mmHg (10-13%)

hepcidin

A protein made predominantly in the liver (low levels are made in macrophages and adipocytes). Hepcidin is produced under the stimulus of inflammatory cytokines and iron excess. When hepcidin is produced it acts to decrease ferroportin, which traps iron in the macrophages. This is a leading mechanism that contributes to the anemia (decrease in the number of functional erythrocytes) that is often seen in the context of inflammatory disease. Ferroportin is also the transport protein used by the intestinal epithelial cells to absorb iron into the blood. Hepcidin also serves to decrease intestinal absorption by decreasing ferroportin (via promoting its destruction) on intestinal cells.

surface-bound immunoglobulins

As the RBC accumulate damage, they become denser and less deformable, largely as the result of repeated rounds of oxidative stress. This leads to accumulation of oxidant injury to hemoglobin, which promotes accumulation of surface-bound immunoglobulins. These immunoglobulins serve as opsonins (more susceptible to phagocytosis) and are one of the main signals for the splenic macrophages to remove older RBCs from circulation.

what is the role of bone marrow adipocytes?

Cell culture experiments have shown marrow adipocytes capable of secreting various cytokines (including IL- 6, G S - CSF and GM- CSF) that can promote proliferation and differentiation of HSCs

iron aquisition by developing RBCs importance

Central to erythropoiesis is the need for cellular coordination of iron acquisition (needed for hemoglobin production) and cell proliferation. A mature RBC must not exit the bone marrow without the proper level of hemoglobin (and hence iron) or it is relatively useless. Small amounts of iron are absorbed in the GI tract daily, whereas the vast majority of iron is recycled from old and dying RBC, into new cells. Macrophages play a central role in this recycling (90- 95% of iron).

why do diabetic patoents develop anemia?

Diabetic patients can develop anemia for a number of reasons. Oxidative damage can increase the turn-over rate or RBC. Also, EPO can become glycosylated as a result of persistently high glucose, and this interferes with the activity of the molecule.

what is the composition of the cytoplasm in mature erythrocytes?

In mature erythrocytes, the cytoplasm is predominantly composed of hemoglobin, with very few organelles. There are essentially no mitochondria in mature RBCs, so they can only utilize glucose anaerobically (glycolysis)

why does inflammation cause nonregenerative anemia?

Inflammatory cytokines skew the differentiation of HSC in the marrow towards myeloid lineages. Inflammatory cytokines induce hepatic synthesis of hepcidin, which as previously described results in iron sequestration a nd a decrease in gastrointestinal absorption of iron. TNF- α decreases EPO expression in the kidney, and also suppresses HIF- 1α signaling in the bone marrow, thus further blunting erythropoiesis in the marrow. Cytokines have been shown to interfere with the progressive development of erythroid cells in culture. For example, IFN - g amma has been shown to cause a down- regulation of the EPO receptor.

how often do hematopoietic stem cells divide?

Long-term HSC are thought to divide once or twice per year, whereas more short term HSC divide on average once per month

what is the result of inflammation on the erythroblastic island?

Macrophages in the erythroblastic island can respond to inflammatory stimuli just as those in other tissue due. In the bone marrow, this can directly impact lineage differentiation and cell proliferation. TNF- alpha causes cleavage of the main transcription factor that drives erythoid differentiation, as well as increasing production of matrix metalloproteinases that can cause disruption in the adhesive interactions between the developing erythoid cells and the stroma or the nurse macrophage.

EPO levels with anemia in renal disease

Most CKD patients (human) have EPO within the reference interval, and sometimes it is even increased relative to the reference interval. However, depending on the degree of anemia, this may be inappropriately low. EPO is approximately the same size as albumin. Therefore, in states where albumin is lost (eg proteinuria) so is EPO. The loss of EPO in renal disease is more a loss of cellular function than it is a parenchymal disease.

what may increased erythropoietic drive indicate in non-anemic animals?

One of the main pathophysiological mechanisms that drives th is is poor oxygenation of the blood, so think cardiopulmonary diseases first. - For example, dogs with mitral valve disease and cardiogenic pulmonary edema frequently present with reticulocytosis without anemia. - brachiocephalic cats have increased reticulocyte counts - diabetes

why are platelets important?

Platelets are important players in hemostasis, immunity, antimicrobial defense, cancer growth and metastasis, angiogenesis, and wound healing.

Erythroferrone (EFN)

Suppresses hepcidin production. EFN basically releases the hold on iron by decreasing hepcidin. EFN will therefore increase intestinal absorption and release of iron from macrophages. EPO drives the production of erythroferrone in the early erythroid precursors (thought to be mostly erythroblasts) in both the bone marrow and spleen.

where are stem cells maintained in a quiescent state?

The area closest to the endosteal surface of the trabecular bone is thought to be where the earliest stem cells are maintained in a quiescent state.

how does the bone marrow respond to changes in demand or tissue injury?

The bone marrow can alter production of cells by a few different mechanisms: - Soluble mediators (cytokines, growth factors) can be secreted from tissue elsewhere in the body that essentially tell the marrow to make more of a certain cell type. This largely occurs when these mediators bind to a very early stem cell and drive it down the selected differentiation pathway. - Alternatively, the signaling event can trigger a faster progression through developmental steps. - The stromal compartment can also be a target, and this is largely by changing adhesion molecules that are holding cells in the marrow or causing the stromal cells to secrete soluble mediators to drive development of HSC. - The marrow compartment itself as well as the surrounding bone are innervated via both the autonomic nervous system and sensory fibers. Part of the interplay between the immune system and nervous system involves what are called inflammatory reflexes. Many of these pathways have been shown to play a role in regulating the egress of cells from the bone marrow. The release of cells from the marrow in response to physiological stress is thought to contribute to the immune response to wounds and sterile inflammation. - Finally, a pathological process occurring within the bone marrow can cause changes in hematopoiesis. Bone marrow is a tissue, and just like any other tissue can be the target of an infectious process, necrosis, and fibrosis.

describe the cell membrame of erythrocytes

The cell membrane is composed of a lipid bilayer with embedded carbohydrates and proteins, and an underlying cytoskeleton. The membrane lipids are important for maintaining cell shape and surface area. The membrane carbohydrates form blood groups, which are predominantly defined by the types of carbohydrates in the red cell membrane. The membrane proteins form various membrane receptors and transport channels. A number of the proteins are linked, both mechanically and functionally, to the metabolism of the RBC. The location of these proteins and affinity for other proteins can be altered by pH within the cell, oxygenation of hemoglobin, and oxidation. Investigators have postulated that some of the "loosening" on the linkages that occurs with deoxygenation of hemoglobin helps to increase the flexibility of the RBC thus potentia lly improving blood flow in hypoxic tissues. This could also lead to membrane damage and "aging" of the RBC.

structure of platelets

The cell membrane of platelets includes a number of glycoproteins that are important in hemostasis. The cytoplasm contains a large number of small granules that often are present in the middle of the cell. This can give a microscopic appearance of a pseudo-nucleus. There are 2 types of granules present within platelets, alpha granules and dense granules. They contain many different molecules that are important for hemostasis.

describe the cytoskeleton of an erythrocyte

The cytoskeleton is critical for maintaining red cell shape and deformability, and is composed of spectrin, actin, band 4.1, ankyrin, and others. These proteins form a complex lattice- like meshwork that allows the RBC to have incredibly high tensile strength as well as deformability. Defects in the cytoskeleton proteins can lead to a loss of structural integrity of the cell, which can shorten its life span or result in visibly recognizable defects in shape

neutrostats

The main "neutrostat" is IL- 17 production by T- ce lls, w h ich w ill drive the production of G- C S F . The predominant "on" signal for this cascade if the GI microbiome. Bacteria in the gut are thought to stimulate innate lymphoid cells to produce first IL- 17, and then G- C S F . IL- 17 production is decreased by polarization of macrophages towards an anti- inflammatory profile. A very potent anti-inflammatory "off" signal to the macrophage is the ingestion of apoptotic cells, especially neutrophils. When these short lived cells are in abundant numbers but dying, this is called efferocytosis, and ultimately quiets down the macrophages, that then suppress T- cell production of G- CSF.

hematopoietic niche

The stroma, osteoblasts, and megakaryocytes of red bone marrow.

what does a high MCHC indicate?

There is no physiologic state where red cells have a higher than normal hemoglobin concentration, so if the MCHC is high, it is usually caused by an artifact, which may be a pathologic or non-pathologic artifact (e.g. red cell agglutination, lipemia).

immunometabolic control of hematopoiesis

This distills down to cells differentiating down different paths, at different rates, dependent upon that totality of the environments they are exposed to. This would include age- related factors, hormones, inflammatory cytokines, nutrient supply. O xygen levels and reactive oxygen species... the list could go on and on! Studies have even show the microbiome in the GI system has an impact on hematopoiesis. The thinking behind immunometabolic control fits with a pathophysiological approach to evaluating bone marrow function.

what is the significance of uremic toxins on erythropoiesis?

Uremic toxins can cause the production of inflammatory cytokines from the endothelial cells , which can also contribute to suppression of erythropoiesis. Some uremic toxins have also been shown to decrease signaling at the EPO receptor within the bone marrow. Some metabolites are derived from bacterial metabolism in the GI tract, can become protein- bound and contribute directly to furthering tissue damage

renin-angiotensin-aldosterone system (RAAS)

a hormone cascade pathway that helps regulate blood pressure and blood volume

steady state hematopoiesis

day-to-day production of a relatively constant number of cells, which occurs largely within the bone marrow

membrane carbohydrates of erythrocytes function

form blood groups, which are predominantly defined by the types of carbohydrates in the red cell membrane

membrane proteins of erythrocytes function

form various membrane receptors and transport channels. A number of the proteins are linked, both mechanically and functionally, to the metabolism of the RBC. The location of these proteins and affinity for other proteins can be altered by pH within the cell, oxygenation of hemoglobin, and oxidation.

hematopoiesis

formation of blood cells from stem cells

macrocytic anemia

high MCV

erythropoiesis is accelerated in response to ___.

hypoxia

what causes hemolytic diseases

immune-mediated RBC destruction (i.e. autoimmune hemolytic anemia), oxidative injury to red cells (e.g. onion toxicity causing oxidative injury and destruction of RBCs), or infectious causes (e.g. Babesia, Mycoplasma). There are also rare hereditary causes of hemolysis, such as G6PD deficiency in horses and PFK deficiency in dogs.

membrane lipids of erythrocytes function

important for maintaining cell shape and surface area

rubriblasts

least mature erythrocytes

hypochromic anemia

low MCHC

microcytic anemia

low MCV

there are three main hematologic manifestations of oxidative damage to RBCs that are visibly detectable are:

methemoglobin, heinz bodies, eccentrocytes

normochromic anemia

normal MCHC

normocytic anemia

normal MCV

when do high levels of intravascular hemolysis occur?

occurs when the RBC is attacked by the complement system (a form of IMHA) or the cells break due to excessive amounts of oxidative or mechanical damage

what does the release of nucleated RBC in absence of the reticulocyte indicate?

pathology

efferocytosis

process by which dying/dead cells (e.g. apoptotic or necrotic) are removed by phagocytic cells

erythropoiesis

production of red blood cells

what is the sequence of maturation of erythroid progenitors within the bone marrow?

rubriblast > prorubricyte > rubricyte > metarubricyte > reticulocyte (polychromatophil) > erythrocyte

where are blood cells derived from?

self-renewing, multipotent hematopoietic stem cells (HSCs)

how is hemolytic anemia identified on microscopic examination?

spherocytes, Heinz bodies, eccentrocytes, or RBC parasites

neutrophilic granulopoiesis stimulation

stimulated by specific hematopoietic grow th factors that are produced within the bone marrow microenvironment by multiple cell types including monocytes, macrophages, endothelial cells, T cells, and bone marrow fibroblasts. These growth factors include SCF, IL- 3, IL- 6, G - CSF, and GM- C S F . They are present in small amounts in healthy individuals, but production increases many-fold in response to inflammatory mediators such as IL- 1β , TN Fα, and LPS . We know that of these grow th factors, G - CSF is crucial in steady- state neutropoiesis, as mice that do not make this protein (via genetic mutation) have a 70- 90% reduction in neutrophil numbers.

what do WBC in the blood reflect?

the balance between production in the bone marrow, release of cells from the marrow compartment, and emigration of the cells into the tissue bed.

granulopoiesis

the production of neutrophils, eosinophils and/or basophils

what do hematopoietic stem cells differentiate into?

various lineages: erythrocytes, myeloid cells, platelets, lymphocytes