Week 1 - Erythropoiesis and the erythrogram Flashcards
Define the term erythron
Definition: the red blood cells and their precursors in the bone marrow.
All erythroid cells in an animal include?
All erythroid cells (red blood cells or RBCs) in an animal
* Includes precursor cells, and RBCs in blood vessels and sinuses in the spleen, liver, bone marrow
What % of an erythrocyte is made up of hemoglobin?
95%
Describe the structure of a hemoglobin molecule.
- Each hemoglobin (Hgb) molecule is a tetramer
- 4 globin chains:
- 2 α-chains & 2 β-chains
- Each globin chain is linked to a
separate heme molecule that
binds O2 - Iron in the ferrous state (Fe +2 ) is
incorporated into each heme
molecule
What is the function of Hgb?
- To transport oxygen (O2) from lungs to tissues
- In health, Hgb is 100% saturated with O2 in arterial blood
How is iron able to transport O2?
- Iron must be in the reduced state (Fe2+ ) to transport O2
- Hemoglobin bound to Fe 3+ cannot carry oxygen
Hgb + Fe 2+ only way to transport O
Some toxins prevent iron from being reduced, so iron will not be transported. Need iron!!
Why is iron important?
- Because hemoglobin synthesis depends on IRON!
- Series of enzymatic reactions for Hgb synthesis
How is iron distributed throughout the body?
- Major sites
1. Erythrocyte Hgb (50-70%)
2. Tissue storage (25-40%) -as ferritin-
3. Remainder in other molecules (myoglobin, cytochromes, enzymes)
How is hemoglobin synthesized?
so won’t have completely formed heme molecule
Describe the condition called Porphyrias. What is it caused by? What does it lead to? What species are affected?
- Rare, hereditary disease in cattle, pigs, cats, & humans
- Caused by deficiency in hemoglobin synthesis enzyme URO-synthase
- Leads to porphyrin accumulation with incomplete heme synthesis
What condition is pictured below?
How would you describe what can be seen in this image?
Porphyria
Marked pink discoloration of the teeth
What condition is pictured below? How would you describe what can be seen in this image?
Porphyria
Bight pink-red fluorescent
color of bone marrow and cartilages viewed under ultraviolet (UV) light.
What are the four major erythrocyte metabolism pathways?
- Embden-Meyerhoff pathway (glycolysis)
- 2,3-DPG (AKA: Rapoport-Luebering) pathway
- Pentose phosphate pathway
- Methemoglobin reductase pathway
Describe the Embden-Meyerhoff pathway (glycolysis)
- Generates ATP to maintain membrane function and
integrity - Generates NADH to reduce methemoglobin
(Fe +3+Hb –> Fe +2+Hb) - Contains 2 clinically relevant enzymes:
- Pyruvate kinase (PK)
- Phosphofructokinase (PFK)
What is the function of the 2,3-DPG (AKA: Rapoport-Luebering) pathway?
Aids in O2 delivery to tissues when anemia (reduced RBC mass) is present
What does the Pentose phosphate pathway generate?
Generates NADPH (cofactor for glutathione reductase)
What is the main function of the Methemoglobin reductase pathway?
Reduces iron (Fe+3 –> Fe +2 )
What is the main purpose of the Pentose phosphate pathway and the methemoglobin reductase pathway?
These 2 pathways provide protection against oxidative stress:
* Toxic exposures can overcome these pathways (more on this later in the semester)
Describe the condition Phosphofructokinase
(PFK) deficiency. What species are affected?
- English Springer Spaniels, American Cocker Spaniels
- Shortened RBC lifespan due to impaired ATP production
- Decreased 2,3-DPG concentration
- Alkalemia-induced hemolytic anemia (hyperventilation when
stressed/excited)
Describe the condition called Pyruvate kinase (PK) deficiency. What species are affected?
- Basenjis, Beagles, Chihuahuas, Dachshunds; Abyssinians, Somalis
- Shortened RBC lifespan
- Accumulation of 2,3-DPG and reduced ATP production
- Bone marrow failure
- Erythropoiesis = erythrocyte production
- Occurs mostly in the bone marrow
Erythropoietin enzyme facilitates this process. Main source of this enzyme is the kidney, sometimes the liver. - Blast-forming unit-erythroid (BFU-E) à committed
stem cells (CFU-E) - CFU-E à rubriblasts (first microscopically recognizable erythroid cell)
- Stimulated by erythropoietin (Epo) and other
cytokines - Epo is produced in adult kidney and fetal liver
Should only see erythrocyte in circulation.
List and describe the erythrocyte development stages.
- Rubriblast
- Prorubricyte
- Rubricyte: basophilic and polychromatophilic
- Last mitotic stage
- Metarubricyte
- Last stage in which the developing RBC is nucleated
- Reticulocyte
- Anucleated
- Larger and more basophilic (more blue) than a mature
erythrocyte – still has stainable cytoplasmic RNA - Mature erythrocyte
- Rubriblast –> erythrocyte
§ Cells produce mRNA for hemoglobin synthesis
§ Cells undergo mitoses to produce more & smaller cells that
have progressively more hemoglobin
§ As hemoglobin synthesis increases in the developing cell, DNA
synthesis decreases and fewer mitotic divisions occur
§ Takes ~ 5 days for erythropoietic progenitor cell –> reticulocyte
§ Reticulocyte is released from bone marrow into circulation and
circulates for 1-2 days before becoming a fully mature
erythrocyte
?
What factors control the process of erythropoiesis?
- Nutritional factors (most important one)
- Hormonal factors
- Bone Marrow factors
Describe the nutritional factors that control erythropoiesis.
- Nucleic acid metabolism
* Vitamin B12 (Aka Cobalamin)
* Folate - Cytoplasmic maturation and hemoglobin formation
* Amino acids
* Vitamin B6
* Iron (critical for Hgb synthesis)
* Copper
Describe the hormonal factors that control erythropoiesis.
- Hormonal factors
- Erythropoietin (Epo) produced in kidney is the main signal for erythropoiesis to occur in the bone marrow.
Ery can also occur in spleen for precursor cells to become mature cells (to replace dead RBC).
Describe the bone marrow factors that control erythropoiesis.
Bone Marrow factors
* Stem cells
* Stroma (if you don’t have enough room for this process to occur).
* Blood supply
What is the lifespan of an erythrocyte?
RBC lifespan in circulation: ranges
* 35 days (birds),
* 70 days (cats),
* 110 days (dogs)
* 145 days (horses)
* 160 days (cows)
§ Importance: may not see anemia right away
How are erythrocytes destroyed?
- Two mechanisms of RBC removal:
§ Major: phagocytosis by macrophages,
mainly in the spleen
§ Minor: intravascular lysis with release of
hemoglobin into plasma
How is Hgb degraded by macrophages?
Excess amount of byproducts –> icterus
What is the main function of Erythron?
- Erythron main function: transport O2 which is
mediated by Hgb - Hbg is transported by erythrocytes
- Hgb consists of heme & globin
Define the term erythrogram.
- Component of a complete blood count (CBC)
- CBC Assesses erythrocytes, leukocytes, and platelets
- Blood is collected into a purple-top tube containing
EDTA and processed through a hematology analyzer - EDTA: anticoagulant
- Chelates calcium to stop the clotting cascade
RBC count is reported in? Measured by?
- RBC (Red blood cell count)
§ Reported in millions
§ Measured by the instrument
Hgb is reported in? Measured by?
- Hgb (Hemoglobin)
§ Typically reported in g/dL
§ Grams of Hgb per 100 mL of blood
§ Measured via spectrophotometry methods
HCT is defined as?
How do you obtain the calculated value?
If HCT is below the RI? Above?
- HCT (Hematocrit)
§ Percentage of blood volume filled by erythrocytes
§ Calculated value (MCV x RBC) / 10
§ If HCT is below the RI, the patient is anemic
§ Usually will have concurrent decreased Hgb and RBC
§ If HCT is above the RI, the patient has an erythrocytosis
- PCV = packed cell volume
- Centrifugal method
- Very accurate with a small error margin
- More accurate than HCT if any RBC parameters are
falsely altered (e.g. MCV, RBC count) - Rule of thumb: HCT and PCV should be ~3x Hgb concentration
- HCT and PCV should differ by maximum of 3%
Erythrogram terminology:
Wintrobe’s indices
* MCV: Mean corpuscular/cell volume
* MCH: Mean corpuscular hemoglobin
* MCHC: Mean corpuscular hemoglobin concentration
MCV is defined as?
How is this value expressed?
How is this value calculated?
If the MCV is below the RI? Above?
MCV: mean corpuscular/cell volume
* Volume per average erythrocyte
* Expressed in fL
* Calculated by some instruments & measured by
others
* (Hct x 10) / RBC
* If MCV is below the RI, there is a microcytosis
* If MCV is above the RI, there is a macrocytosis
MCH is defined as?
How is this value expressed?
How is this value calculated?
If the MCH is below the RI? Above?
MCH: mean corpuscular hemoglobin
* Quantity of Hgb per average erythrocyte, expressed
in picograms (pg)
* Calculated value
* (Hgbx10)/RBC
* If MCH is below the RI, there is hypochromasia
* If MCH is above the RI, there is hyperchromasia
Inaccuracies in RBC count will lead to?
Inaccuracies in RBC count
will lead to
MCV and MCH inaccuracies
MCHC is defined as?
How is this value expressed?
How is this value calculated?
If the MCHC is below the RI? Above?
MCHC: mean corpuscular hemoglobin concentration
* Cellular Hgb concentration per average erythrocyte
* Expressed as grams of Hgb per 100 mL erythrocytes
(g/dL)
* Calculated value
* (Hgbx100)/Hct
* More accurate than MCH
* If MCHC is below the RI, there is hypochromasia
* If MCHC is above the RI, there is hyperchromasia
What can influence MCV and MCHC values?
Pre-analytical factors
can influence MCV and MCHC values
* Short sample (underfilled EDTA tube) or low plasma
osmolality (low Na and Cl)
- use correct size tube for blood/sample so that machine can run correctly
* Falsely decreased MCV and increased MCHC
* Increased plasma osmolality (high Na and Cl)
* Falsely increased MCV and decreased MCHC
What does it mean when a patient has a low number of Reticulocytes? High number? What is the exception to this rule?
- In health, low numbers normally present in
circulation in most species - When increased, indicates increased RBC
production (regenerative anemia) - Typically interpreted in context of an anemia
(regenerative or non- regenerative) - *Exception: horses release few (if any) reticulocytes
- Cattle and small ruminants: basophilic stippling
Make sure machines are calibrated to the proper species due to differences in cell morphology, etc.
How do you count reticulocytes?
- Instrument uses a special dye for reticulocyte RNA and measures what percentage of all erythrocytes are reticulocytes
- Can also manually count on a blood smear stained with new methylene blue
How do you obtain the absolute reticulocyte count (absRC).
- absRC = reticulocyte percentage x RBC count (in millions)
- Reported by some hematology analyzers
- Can be calculated
How do you obtain the corrected reticulocyte percentage (CRP)?
- CRP = reticulocyte% x (patient’s HCT/normal HCT for that species)
- Normal HCT used for
§ canines = 45%
§ Felines = 35%
- How are nucleated RBC’s (nRBCs) typically expressed?
- The number of nRBC’s can be increased in what situations?
- nRBC’s are usually?
Do not see them often. May see a couple or a few.
* Typically expressed as number per 100 WBCs
counted
* See an increased number in these situations:
* strongly regenerative anemias or
* damage to bone marrow endothelium
* Usually metarubricytes or rubricytes, but can
include earlier stages in very strongly regenerative
anemias or leukemia
What are the arrows pointing to in this image?
Reticulocytes
What is the arrow pointing to in this image?
nRBCs
- What does the red cell distribution width (RDW) represent?
- How do you obtain the calculated value?
- What does RDW indicate?
- Is the RDW indicated by all analyzers?
- Represents coefficient of variation of RBC volume
distribution - Calculated value: RDW = (SDMCV/MCV)*100
- Indicates degree of anisocytosis (variation in RBC
size) - Not reported by all analyzers
- What does plasma protein represent?
- How is plasma protein determined?
- What is it interpreted with?
- If the plasma protein is above the RI? Below?
- Plasma protein (aka TP or “total solids”)
- Reported as g/dL
- Determined by refractometry
- Usually interpreted in conjunction with RBC parameters
- If below the RI, indicates Hypoproteinemia
- By loss or lack of production
- If above the RI, indicates Hyperproteinemia
- By increased production or hemoconcentration
- How is fibrinogen measured?
- Is this routinely reported?
- If fibrinogen is below the RI? Above?
- Measured in large animals by heat precipitation method.
- Not routinely reported on a CBC in small animals
* If below the RI, it is called Hypofibrinogenemia
* indicates loss or lack of production
* If above the RI, it is called Hyperfibrinogenemia
§ Indicates increased production (inflammation)
§ Considered Positive acute phase protein
§ indicates hemoconcentration
- Not routinely reported on a CBC in small animals
Explain the two ways in which you can calculate the absRC.
high MCV = macrocytic
MCH high and MCHC is high = macrochromic BUT even though MCH is flagged, the MCHC is more accurate. Th
Macrocyctic normochromic anemia?
9 x 1.2 = 10.8
low MCV = microcytic
other params norm = normochromic
norm MCV = normocytic
MCH low = microchromic
normocytic microchromic
