1. MLAB 1315- Hematology Keri Brophy-Martinez
Chapter 5: The Erythrocyte

2. Erythrocyte Maturation
Erythropoiesis
Production and maturation of erythrocytes
Erythropoietin (EPO)
The growth factor that stimulates RBC production
Released in response to decreased levels of oxygen in the body tissues
Hormone produced and released by the kidneys which acts on committed RBC stem cells to stimulate red cell maturation and release into the blood
With normal levels of EPO stimulation and normal red cell lifespan, about 1% of the red cells in the blood are newly released red cells called reticulocytes. Aged rbc’s are primarily removed by the spleen.
Deficient O2 delivery to the tissues causes the kidney to increase EPO release to accelerate red cell production

3. Normal RBC in adults Male 4.7 - 6.1 x 106/µl Female 4.2 - 5.4 x 106/µl
Infants and children - normals vary by age

4. Maturation Sequence of Erythrocytes
Stem cell - an unspecified cell that gives rise to a specific specialized cell, such as a blood cell
Multipotential and cannot be identified morphologically
Can self-renew and differentiate
CFU-GEMM: granulocyte, erythrocyte, monocyte, megakaryocyte
BFU-E: burst forming unit
CFU-E: colony forming unit
EPO

5. Maturation Sequence of Erythrocytes
Rubriblast (Pronormoblast)
Size = µm
Cytoplasm
Deeply blue (basophilic
Scant amount, may have a perinuclear halo
No granules
Nucleus
Large and round
Reddish-purple with fine chromatin
1-2 nucleoli (may be bluish)
N:C ratio ( nuclear: cytoplasmic)= 4:1

6. Maturation Sequence of Erythrocytes
Prorubricyte (basophilic normoblast)
Size = 10-16µm
Cytoplasm
Deeply basophilic indicating RNA activity needed to produce hemoglobin (no hemoglobin is present at this stage)
No granules
Nucleus
Round, large
Chromatin more clumped
No nucleoli
N:C ratio = 4:1

7. Maturation Sequence of Erythrocytes
Rubricyte (polychromatic normoblast)
Size = 10-12µm
Cytoplasm
Blue-gray to pink-gray (pink indicates that hemoglobin production has begun)
Slight increase in amount
Nucleus
Round and smaller
Chromatin more clumped, irregular
No nucleoli
N:C ratio = 4:1

8. Maturation Sequence of Erythrocytes
Metarubricyte - Nucleated RBC (orthochromic normoblast)
Size: µm
Cytoplasm
Pinker indicating larger amounts of hemoglobin production
Increased amount
Nucleus
Tightly condensed chromatin (pyknotic)
No nucleoli
Mitosis ends at this stage (no more DNA synthesis)
Nucleus is extruded at end of this stage
N:C ratio = 1:1

9. Maturation Sequence of Erythrocytes
Reticulocyte (diffusely basophilic or polychromatophilic erythrocyte
Size: 8-10µm
Cytoplasm
Diffusely basophilic due to residual RNA
Stain with new methylene blue to see fine reticulum strands
Hemoglobinization is not complete
No nucleus present
Present in circulation for 1-2 days

10. Lab Methods
New Methylene Blue is a supravital stain it is used to stain reticulocytes. They cannot be identified as reticulocytes from Wright’s stain.

11. Maturation Sequence of Erythrocytes
Mature erythrocyte
Size = 7-8µm
Volume = fL
Cytoplasm
Pink/red
Biconcave shape
Nucleus - none
Present in circulation for about 120 days

12. Red Blood Cell Membrane
Development
Trilaminar, three-dimensional structure
Outermost layer: glycolipids, glycoproteins
Central layer: cholesterol, phospholipids
Inner layer: cytoskeleton
spectrin
Composed of alpha & beta chains
Join to form a matrix which strengthens the membrane against sheer force and controls biconcave shape
ankrin
membrane proteins

13. Red Blood Cell Membrane
Function
Shape
Provides the optimum surface to volume ratio for respiratory exchange AND is essential to deformability
Provide deformability, elasticity
Allows for passage through microvessels
Provides permeability
Allows water and electrolytes to exchange
RBC controls volume and H2O content primarily through control of sodium and potassium content

14. Red Blood Cell Metabolism
These pathways are essential for oxygen transport and maintaining the physical characteristics of the RBC.
Embden-Meyerhof glycolytic pathway
Generates 90% of energy needed by RBC’s
Glucose is metabolized and generates two molecules of ATP (energy).
Hexose monophosphate shunt
Metabolizes 5-10% of glucose.
NADPH is end product
Protects the RBC from oxidative injury.
Most common defect is deficiency of the enzyme glucose-6-phosphate dehydrogenase (G-6PD).
If the pathway is deficient, intracellular oxidants can’t be neutralized and globin denatures then precipitates. The precipitates are referred to as Heinz bodies. (Must use supravital stain to visualize them.)

15. Red Blood Cell Membrane
Methemoglobin reductase pathway
Maintains iron in the ferrous (Fe2) state.
In the absence of the enzyme (methemoglobin reductase), methemoglobin accumulates and it cannot carry oxygen.
Leubering-Rapaport shunt
Allows the RBC to regulate oxygen transport during conditions of hypoxia or acid-base imbalance.
Permits the accumulation of 2,3-DPG which is essential for maintaining normal oxygen tension, regulating hemoglobin affinity

16. Checkpoint
Which erythrocyte metabolic pathway is responsible for providing the majority of cellular energy?
For regulating oxygen affinity?
For maintaining hemoglobin in a reduced state?

17. Checkpoint Embden-Meyerhof :90-95%
Rapoport-Leubering shunt: oxygen affinity
Hexose-Monophosphate shunt/ Methemoglobin Reductase pathway: iron

18. Red Blood Cell Metabolism: Summary
Three Areas of RBC metabolism are crucial for RBC survival and function.
RBC membrane
Hemoglobin structure and function
RBC metabolic pathways= cellular energy

19. Erythrocyte Destruction
Breakdown of the RBC
Toward the end of 120 day life span of the RBC, it begins to break down. This is about 1% of RBC’s per day.
The membrane becomes less flexible.
The concentration of cellular hemoglobin increases.
Enzyme activity, especially glycolysis, diminishes
Aging RBC’s or senescent RBC’s are removed from the circulation by the reticuloendothelial system (RES) which is a system of fixed macrophages. These cells are located all over the body, but those in the spleen are the most efficient at removing old RBC’s.

20. Extravascular hemolysis
90% of RBC’s are destroyed extravascularly.
Occurs in splenn, liver and bone marrow
The RES cells lyse the RBC’s and digest them. Components of the RBC are recycled.
Iron is transported by transferrin to the bone marrow to be recycled into hemoglobin.
Amino acids from globin are recycled into new globin chains.
The protoporphyrin ring from heme is broken and converted into biliverdin (green).
Biliverdin is converted to unconjugated bilirubin and carried to the liver by albumin, a plasma protein.
Bilirubin is conjugated in the liver and excreted into the intestine, where intestinal flora convert it to urobilinogen.
Most urobilinogen is excreted in the stool, but some is picked up by the blood and excreted in the urine.
Conjugated (indirect) and unconjugated (direct) bilirubin can be used to monitor hemolysis.
Refer to pg.65

21. Intravascular hemolysis
5-10% of RBC’s are destroyed intrasvascularly
RBC breakdown occurs within the blood vessels.
The free hemoglobin α and β dimers that are released into the bloodstream is picked up by a protein carrier called haptoglobin.
The haptoglobin-hemoglobin complex is large and cannot be excreted in the urine. It is carried to the liver where the RES cells and the breakdown process occurs as above.
If there is an increase in intravascular hemolysis, the haptoglobin is used up and free hemoglobin is excreted in the urine (hemoglobinuria).
Free hemoglobin may also be oxidized to methemoglobin which is then broken down extravascularly or to methalbumin which is bound to albumin
Refer to page 66