Harvey Chapter 4 Evaluation of Erythrocytes PART I AKG Flashcards
Esterified or non-esterifide cholesterol increases fluidity of the RBC membrane?
Esterified - it is builker and ‘packs’ more loosely
How does the degree of saturation of phospholipid fatty acids affect RBC membrane fluidity?
More saturation –> better ‘packing’ –> decreases fluidity of the membrane
What is the predominant cytoskeletal protein within RBCs?
Alpha and beta sepctrin
This cytoskeletal RBC protein:
- binds spectrin along with protein 4.1
- involved in junctional complexes that link cytoskeleton to cell membrane
Actin
This cytoskeletal RBC protein:
- part of actin junctional complex (binds glycophorins and band 3)
- Binds both ß-spectrin and actin to strengthen the spectrin-actin interaction
Protein 4.1
This cytoskeletal protein attaches ß-spectrin to band 3 tetramers.
Ankyrin
What is the integral membrane protein responible for:
- (1) anchoring cytoskeleton to the membrane
- (2) anion exchange (delivering CO2 to the lungs in exchange for Cl-)
- (3) Binding of glycolytic enzymes, hemoglobin and hemichromes
Band 3 (“anion exchange protein”)
Which integral membrane protein is heavily glycosylated and responsible for:
- Carrying most of the sialic acid residues, and therefore negative charge on the surface of RBCs
Glycophorin
Each RBC contains approximately ____ hemoglobin molecules.
A. 2.5 billion
B. 250 million
C. 250,000
D. 25
B. 250 million
Describe the structure/composition of hemoglobin
- Hemoglobin is a tetramer:
- Two α- and two β-polypeptide globin chains (4 globins total) + 4 heme proteins.
- Each globin is linked to a separate heme that binds oxygen
- The heme prosthetic group is held firmly within a hydrophobic cleft
- Non-polar side of heme is positioned in the hydrophobic pocket of each globin chain
α-globulins in hemoglobin bind and transport _____
ß-globulins in hemoglobin binds _____
CO2
2,3-DPG
List the steps of heme synthesis and where they occur within the cell.
Mitochondria –> cytosol –> mitochondria
- Mitochondria: glycine + succinyl CoA –5-ALA synthase –> delta-ALA
- Cytosol: delta ALA –porphobilinogen synthase–> porphobilinogen –> protoporphyrin IX
- Mitochondria: Protoporphyrin IX + Fe2+ –ferrochelatase enzyme–> heme
List the enzymes that are inhibited by lead in heme synthesis.
What are thhe subsequent consequences?
- 5-ALA synthase
- Porphobilinogen synthase
- Ferrochelatase
- Coproporphyrinogen oxidase
Build up of porphyrins in RBCs, plasma, and/or urine (aka porphyria) - these animals are prone to photosensitivtiy.
Early in hemoglobin synthesis, 5-ALA synthase requires a co-factor to make delta-ALA. What is this cofactor?
B6 (pyridoxine)
How is free hemoglobin as a result of IVH normally cleared?
Binds to haptoglobin and the complex is cleared by macrophages
If free hemoglobin exceeds binding capacity of haptoglobin, hemoglobin is cleared by the kidneys (hemoglobinuria)
Heme catabolism starts with cleavage of the heme ring mediated by ______ which releases linear tetrapyrrole biliverdin, iron, and carbon monoxide. This enzyme is highest in which organ?
heme oxygenase
it’s activity is highest int he spleen; some ativity in the liver, BM, and renal tubular cells
During heme catabolism, once iron is released, it is oxidized to the ____ form and is released and trasnported as ______ and stored as ____ or ____ in the liver and BM for subsequent reuse.
ferric
transferrin
hemosiderin or ferritin
After biliverdin is released from cleavage of the heme ring mediated by heme oxygenase that occurs during heme catabolism, list what happens to biliverdin next in mammals vs birds.
Mammals:
- Biliverdin reduced to bilirubin within macrophages via bilirubin reductase
- Bilirubin binds to albumin to remain soluble in plasma and the complex is transported to the liver
- Hepatocyte uptakes bilirubin and conjugates it to glucuronic acid via glucuronyl transferase
- Hepatocytes transport bilirubin glucuronide into bile canaliculi –> rate limiting step. (small amount is released into circulation)
Birds:
- Birds lack biliverdin reductase, so avian bile contains biliverdin - very green bile compared to yellow/green bile in mammals
Sheep and goats synthesize hemoglobin ___ in response to severe anemia (mediated by EPO).
hemoglobin C
What are the two hemoglobin confirmations? Where do they mostly occur within the body?
What is the relationship between the confirmations and oxygen affinity and 2,3-DPG affinity?
- R (relaxed): lungs
- High O2 affinity
- Low affinity for 2,3-DPG
- T (tense): metabolically active tissue
- Low O2 affinity (deoxyHgb)
- High affinity for 2,3-DPG
2,3DPG is an organic phosphate that binds to hemoglobin and ____\_ oxygen-binding affinity of hemoglobin in RBCs.
decreases
2,3-DPG is highest in which species and lowest in which?
- Highest in: rabbits, pigs, horses rats
- Moderate in: humans and dogs
- Very low in: cats and ruminants
- Low to absent in birds (birds can use insoitol pentaphosphate as an alternative to 2,3 DPG)
Hypophosphatemia and acidosis ___ 2,3-DPG synthesis
Increases in [phosphate] and alkalemia (e.g., hypoxia - respiratory alkalosis) ____ 2,3-DPG synthesis.
decrease
increase
Define the Bohr effect
A decrease in hemoglobin oxygen-affinity in response to a lowered blood pH resulting from an increased concentration of CO2 in the blood.
*so hemoglobin oxygen binding affinity is inversely related to aciditiy (H+) and CO2. Acidity and/or high CO2 in blood (as seen in metabolically active tissues) shifts the oxygen-hemoglobin dissociation curve right (promote T conformation to let go of oxgyen). Conversely, low CO2 and higher pH (as seen in the lungs), shifts the O2-hemoglobin dissociation curve to the left (promote R conformation - to hold onto oxygen).
Please draw the oxygen-hemoglobin dissociation curve. Label the x and y axis, and draw and describe what drives the curve to shift to the left and right (DPG, pH, CO2, temp).
What are the three man functions of erythrocytes?
- Transport O2 to tissue
- Transport CO2 to the lungs
- Buffering hydrogen ions (Deoxyhemoglobin is a weaker acid than oxyhemoglobin → when oxyhemoglobin releases O2 in tissues, the formation of deoxyhemoglobin results in increased binding of H+, buffering the effects of H2CO3 and allows for the isohydric transport of CO2)
Which species have high intracellular potassium d/t high Na/K ATPase activity?
Horse
Pig
Some ruminants
Japanese and korean dogs
Some dogs have increased glutamate tarnsport, which results in high reduced glutathione (GSH) concentrations. This promotes the replication of which organism within RBCs?
Babesia gibsoni
What is the primary substrate for energy needs of all RBCs in mammals? What is the exception?
Glucose
Except pig RBCs, which use inosine.
Pig RBCs are poorly permeable to glucose because they lack functional glucose transporter.
Glucose enters RBCs and is phosphorylated to G6P via hexokinase. It can then either be metabolized via ____ pathway or ____ pathway.
Embden-Meyerhof pathway (anaerobic glycolysis)
Pentose phosphate pathway
Mature RBCs undergo kreb’s cycle for energy. T/F
False - they use embden-meyerhof pathway (anaerobic glycolysis) becuase they do not have nucleus, ribosomes, mitochondria, and ER.
____ blood inorganic phosphate concentration and ____ blood pH stimulate anaerobic glycolysis via EMP.
↓ or ↑
↑
Hemolytic anemia in diabetic dogs and cats and post-parturient cattle and a cat with hepatic lipidosis results from decreased ______ which decreases RBC glycolic rates and ultimately decreases ATP generation.
Phosphorous (hypophsophatemia)
Methemoglobin is formed when hemoglobin iron is oxidized from the ___ to the ___ state.
ferrous (2+) to the ferric (3+)
NADPH generated by the ____ in RBCs provides electrons to protect against oxidants by maintaining glutathione and thioredoxin in their reduced states.
Pentose phosphate pathway (PPP)
About 3% of hemoglobin (Fe2+) is oxidized to methemoglobin (Fe3+) each day; methemoglobin is reduced back to hemoglobin in a reaction requiring both __________ and _______ generated by anaerobic glycolysis.
cytochrome-b5 reductase and NADH
How is iron distributed in the body?
- 60-70% of total body iron is present in hemoglobin
- 1/3rd is stored as ferritin and hemosiderin within macrophages
- only 0.1% is bound to transferrin in plasma.
This molecule is expressed in the enterocytes of the villi and not in the crypts; also expressed on vesicle membranes inside erythroid precursors; transports iron from the enterocyte to the circulation, and from endosomes of macrophages to the cytoplasm; will increase with an iron-deficient diet and decrease by an iron replete diet
Divalent Metal Iron Transporter-1 (DMT-1)
This molecule acts as a reductase enzyme, reducing dietary ferric iron (Fe3+) to ferrous iron (Fe2+) so that it can be transported by DMT-1 from the lumen and into the enterocyte; located on the apical membrane of the enterocyte
Ferric reductase (aka Duodenal Cytochrome b)
This molecule is a copper-dependent transmembrane iron oxidase; expressed in the small intestine in the basolateral membrane of enterocytes; responsible for oxidizing ferrous iron (Fe2+) to ferric iron (Fe3+) so that it can be transported out of the enterocyte and into plasma
Hephaestin
This molecule is expressed on the membrane of duodenal enterocytes (basolateral side), placental trophoblasts, and macrophages distributed in various tissues; the only mammalian iron exporter out of cells (exports iron from enterocytes into circulation and from macrophages into circulation); expression levels regulated by hepcidin binding, causing internalization of the protein and by iron levels
Ferroportin
- This molecule is composed of 24 protein subunits arranged in a spherical shell that contains pores for iron entrance and exit
- it is a storage protein that stores excess iron in the cell
- prevents free iron from catalyzing oxidative reactions which would injure the cell
- has a plasma form and a tissue form
- it is a positive acute phase protein
Apoferritin - when complexed with iron, it is called ferritin
This molecule results from degradation of ferritin, whereby the apoferritin protein shell has been removed; composed of aggregates of protein and iron within lysosomes
Hemosiderin
This molecule is a protein/hormone secreted by hepatocytes into circulation; regulates iron export from enterocytes and macrophages by causing internalization and lysosomal degradation of ferroportin
Hepcidin
Things that upregulate hepcidin:
- Increased iron stores
- BMP-SMAD
- Hemojuvelin (HJV)
- Inflammatory cytokines (IL-6)
- JAK-STAT
- BMP-SMAD
Things that suppress hepcidin:
- Erythropoietic reactivity
-
Erythroferrone (ERFE)
- Produced by erythroid precursors
- Upregulated by EPO
- Stimulates iron absorption and release
- Facilitates iron delivery during stress erythropoiesis
- Contributes to iron overload in ineffective erythropoiesis
-
Erythroferrone (ERFE)
- Hypoxia and iron deficiency
- TMPRSS6 (Matriptase-2)
- Suppresses hepcidin by cleaving hemojuvelin (HJV)
- Produced mainly by the liver
- Mutation -> iron-refractory iron deficiency anemia (IRIDA)
- High hepcidin
- Iron present in enterocytes, but high levels of hepcidin prevents much from getting to erythroid precursors (aka secondary iron deficiency)
- TMPRSS6 (Matriptase-2)
Refractory to oral or parenteral iron
This molecule is produced by erythroid precursors and is upregualted by EPO. It stimulates iron absorption and release - it facilitates iron delivery during stress erythropoiesis and contributes to iron overload in ineffective erythropoiesis. It suppresses hepcidin.
Erythroferrone (ERFE)
This molecule is produced mainly by the liver and suppresses hepcidin by cleaving hemojuvelin (HJV).
TMPRSS6 (Matriptase-2)
Mutation in TMPRSS6 (Matriptase-2) leads to ______ due to high levels of hepcidin in the body.
iron-refractory iron deficiency anemia.
Mutations in HJV, TFR2, or HFE result in too little hepcidin and therefore ______.
hemochromatosis
This molecule is formed by iron binding to apotransferrin; serves as a transport protein and delivers iron to RBCs and prevents free iron-catalyzed oxidative reactions
transferrin
______ is a major copper-carrying protein in blood and plays a role in changing iron from ferrous (2+) to ferric (3+) state before it binds to transferrin.
______ is a transmembrane, copper-dependent ferroxidase protein found on the basolateral membrane of enterocytes that is responsible for transporting dietary iron from intestinal enterocytes into the circulatory system
Ceruloplasmin
Hephaestin
What is the average life span of RBCs?
2-5 months
This is a protein that binds free heme in plasma and once bound forms a complex that is recognized by macrophages and hepatocytes; once recognized, heme undergoes endocytosis and the cell membranes are protected from toxic effects of free heme.
Hemopexin
This RBC abnormality is common in iron-deficient ruminants (including llamas and alpacas).
Dacrocytes
Name the RBC abnormality and its significance.
Torocytes
Generally artifacts.
Rouleaux is normal in which species? Why does this occur?
What potentiates rouleaux formation?
- Horses, cats, and pigs
- RBCs that have less negative charge on surface (weaker electrostatic repulsive force) aggregate more readily than cells with opposite characteristics
- Increased concentrations of globulins (fibrinogen, haptoglobin, immunogloblin) - as may be associated with inflammation or lymphoproliferative disorders, potentiates rouleaux formation
Which Ig has the greatest propensity to produce agglutation?
IgM due to it’s pentavalent nature
Poikilocytosis may be normal in which species?
Goats and young cattle
Echinocytes form when surface area of outer lipid monolayer increases relative to inner monolayer. What is the significance of echinocytes (‘crenated’ RBCs) on a blood smear?
- Usually artifact d/t excess EDTA, smear preparation or prolonged storage
- May occur in presence of fatty acids, lysophospholipids, and amphipathic drugs that distribute preferentially in outer half of lipid bilayer
- May form when RBCs are dehydrated, pH is increased, ATP is dpeleted, and intracellular calcium is increased
Acanthocytes form when RBC membranes contain excess ____ compared to phospholipids.
cholesterol
What is the most common cause of stomatocytes on blood smear?
What are other causes?
- Artifact due to thick film preparation
- Can form when blood pH is decreased and amphiphatic drugs that distribute preferentially in inner leaflet of lipid bilayer (opposite of echinocytes); or when water content of RBCs is increased
- Hereditary stomatocytosis
Spherocytes with slight indentations on one side may be called _____.
stomatospherocytes
These are thin, often hypochromic-appearing RBCs with increased membrane-to-volume ratios. They can be folded. They can appear as triconcave knizocytes and may appear as codocytes (‘target cells’). Codocytes are especially increased in dogs with congenital _____. ______ may be seen in iron deficiency anemia.
Leptocytes
Dyserythropoiesis
- This RBC morphological abnormality results from oxidant damage to RBC membranes which leads to adhesion of opposing areas of cytoplasmic face of RBC membrane (think dumplings/pierogi)
Eccentrocytes
- This RBC morphological abnormlaity appears as an irregularly spherical erythrocytes with small cytoplasmic projections.
- Product of oxidant injury; may develop from eccentrocytes following loss of much of the fused membrane
- Have higher hemoglobin concentrations than normal and represent RBCs undergoing eryptosis
Pyknocyte
This RBC morphology is normal in Camelidae family and other exotics. It is recognized abnormally in cats with bone marrow abnormalities (myeloid neoplasm and acute lymphoblastic leukemia), hepatic lipidosis, portosystemic shunts, doxorubicin toxicity, dogs with meylofibrosis, myelodysplastic syndrome, and glomerulonephritis
Elliptocyte (ovalocyte)
This RBC morphology is observed in blood of normal deer. It may develop secondary to hemoglobin polymerization (which occurs normally within tubular filaments in adult Angora goats)
Drepanocyte
What are these and what is their significance?
EM of hemoglobin crystals
Can be seen in horses with FAD and G6PD deficiency
Frequently found on blood of llamas and alpacas, occasionally in cats
Unknown mechanisms of formation
What are these and what are their significance?
Erythroid loops
Found in dogs bitten by the asp viper
___ are RBC microvesicles that form following inward budding of membranes to form multivesicular bodiees, which then fuse with external cell membrane and are released. This process is used by reticulocytes to eliminate uneeded proteins .
_____ are RBC microvesicles that are shed directly from the cell membranes and are released during conditions that result in eryptosis.
Exosomes
Ectosomes
- Removed/”pitted” by the spleen as reticulocytes and erythrocytes squeeze through interendothelial slits of the splenic sinus
- Present normally in low numbers in RBCs of horses and cats; present in regenerative anemia or in patients post-splenectomy; increase in animals with glucocorticoid therapy, in poodle macrocytosis, and animals tx with chemo
- Stain dark blue
Howell-Jolly bodies
- large aggregates of oxidized, precipitated hemoglobin attached to internal surfaces of RBC membranes
- Stain red to pale pink with Romanowsky-type stain; stain lighter blue than
- Normal cats may have up to 5%; abnormal to see in any other species – indicate oxidative damage – endogenous or exogenous
Heinz bodies
- occurs when ribosomes aggregate together, forming blue-staining punctate inclusions
- commonly occurs in regenerative anemia in ruminants
- Prominent in any species in the presence of lead poisoning
Basophilic stippling
- Iron positive inclusions
- Generally focal, basophilic inclusions located near periphery of RBC
- May consist of cytoplasmic ferritin aggregates and/or iron-loaded mitochondria
- Normal to see ferritin aggregates in nucleated RBCs of humans, dogs, and pigs
- Presence of iron-loaded mitochondria is a pathologic finding in all species
- Prussian blue staining is used to verify presence of iron-positive material
- Can be seen in disorders of mitochondrial iron metabolism, administration of chloramphenicol, lead, hydroxyzine, zinc, and oxazaolidinone antibiotic
siderocytes
What is this and what is its significance?
Reddish purple staining threadlike loop/figure 8 structure that can be observed in normal camel and llama RBCs stained with reticulcoyte stains (primarily found in reticulocytes in humans)
name organism in dog
babesia canis
Name organism in dog
Babesia gibsoni
name organism in horse
Babesia equi
name the organism (left is cow, right is deer)
Theileria
name the organism in a cat
Cytauxzoon felis
Name the organism, cow
Anaplasma marginale
Name the organism. EM of bovine erythrocyte.
Anaplasma marginale
Note the binary fission of the organism within an erythrocyte vacuole in A, and six organisms within erythrocyte vacuole in B.
Name the organism. Dog
Distemper inclusions
Name the organism. Cat. EM of erythrocytes.
Mycoplasma haemofelis
Name the organism. Cow. RBC EM.
Mycoplasma wenyoni
Name the organism. Transmission photomicropagh of cat RBC.
Mycoplasma haemofelis
Name organism.
E. Pig
F. Sheep
G. Cow
H. LLama
E. Pig: M. suis
F. Sheep: M. ovis
G. Cow: M. wenyoni
H. LLama: M. haemolamae
Small rod-shaped bacterium occurs within erythrocytes in cats. Name the organism.
Bartonella henselae
What are the two major reducing agents in RBCs that are used to maintain hemoglobin and other proteins in a functional reduced state?
NADPH and GSH
