Hematology Solo 3 Erythrocytes Lecture 1 Erythropoiesis, Maturation, Membrane Characteristics, and Metabolic Activities

Erythropoiesis Nomenclature

Pronormoblast (Rubriblast)

Basophilic Normoblast (Prorubricyte)

Polychromatophilic Normoblast (Rubricyte)

Orthochromic Normoblast (Metarubricyte)

Polychromatophilic Erythrocyte (Reticulocyte)

Mature Red Blood Cell (Mature Erythrocyte)

Reticulocytes are only classified as such when stained with new methylene blue revealing organelles in a granulofilamentous arrangement

Erythropoiesis

The production of erythrocytes in the bone marrow.

This occurs in erythropoietic islands of medullary bone marrow over a period of about 5 days.

The average life span of a maturing red blood cell is 120 days

Erythropoietic Islands

Distinct anatomic units that consist of a macrophage surrounded by a cluster of maturing erythroblasts

Hgb synthesis occurs

Arrow is pointing to a macrophage

Erythropoiesis

Begins with a pluripotential stem cell followed by a progenitor cell, which is stimulated by Erythropoietin (EPO) for erythrocyte production

EPO is a hormone produced by the kidney that maintains a constant red cell mass

1. 1. Renal sensors sense a decrease in the body’s oxygen level

2. 2. EPO production is increased

3. 3. This stimulates the bone marrow erythroid precursors to proliferate and increase the production of red blood cells

Disorders Related to Erythrocyte Maturation and Production

Disorders of Erythropoietin

Polycythemia is the term used to refer to an increased concentration of erythrocytes (erythrocytosis) in the circulating blood that is above normal for gender and age

Secondary, or absolute, polycythemias reflect an increase in erythropoietin production and should not be confused with polycythemia vera or relative polycythemias

Mechanisms that can produce secondary polycythemia include the presence of high oxygen affinity hemoglobin, chronic lung disease, smoking, and dwelling in high altitudes

RBC Maturation: Key Points

The stage of maturation is determined by

1. 1. Cell size

2. 2. Nuclear size compared to the rest of the cell: Nucleus to cytoplasm (N:C) ratio

3. 3. Nuclear chromatin: Texture, density, homogeneity

4. 4. Presence or absence of nucleoli

5. 5. Color of cytoplasm

Nucleus is round in all maturation phases

The primary characteristic to evaluate when determining maturation is Nuclear Chromatin

Erythropoiesis

The following changes occur in the developmental stages of RBC maturation

1. 1. Cell volume decreases

2. 2. N:C ratio decreases

3. 3. Nuclear chromatin goes from fine to becoming condensed

4. 4. Nucleoli disappear

5. 5. Cytoplasm color changes from blue to pink as RNA decreases and hemoglobin synthesis increases

Select the correct maturation sequence of erythropoiesis

Rubriblast, Prorubricyte, Rubricyte, Metarubricyte, Diffusely basophilic erythrocyte, Erythrocyte

As erythroid cells mature with each stage

Amount of RNA in the cytoplasm decreases

Rubriblast

Also known as pronormoblast or proerythroblast

Earliest and largest cell in the erythroid series

Cell size is 14-24 micrometers

Nucleus

• Nucleus is round (almost spherical) with distinct, linear chromatin strands

• Nuclear to cytoplasmic ratio is high (8:1 to 6:1)

• Nucleus stains reddish-blue with 0-5 nucleoli visible

• Chromatin: blue, fine, diffuse, loose, no evidence of clumping

Cytoplasm is deep royal blue; scant amount with no granules

Reference range: 0-1.5% in bone marrow

Prorubricyte

Also known as basophilic normoblast, basophilic erythroblast

Cell size is 12-17 micrometers

Nucleus

• Round shape, reddish color with slightly coarse chromatin

• N:C ratio is high: 6:1-4:1

• Nucleoli is difficult to see or absent; 0-1 present

Cytoplasm is basophilic due to clusters of free ribosomes and RNA; slight amount

Reference range: 1%-5% found in bone marrow

Rubricyte

Also known as polychromatophilic normoblast or polychromatophilic erythroblast

Cell size is 10-15 micrometers

Nucleus

• Dark blue, smaller, coarse and clumped

• N:C ratio is moderate: 4:1 to 2:1

• Nucleoli is no longer visible

Cytoplasm contains various shades of pink and blue; moderate amount with no granules

Reference range: 5-30% of cells in normal bone marrow

Lymphocyte (left) vs rubricyte or polychromatophilic erythroblast (right)

Metarubricyte

Also known as orthochromatic normoblast, orthochromatic erythroblast

Cell size is 8-12 micrometers

Nucleus

• Round, usually eccentrically located

• N:C ratio is 1:1-1:2

• Chromatin: small, pyknotic, condensed, no parachromatin (white areas in the nucleus), completely black appearance, no nucleoli present

• Nucleus is extruded during the later stages of the metarubricyte

Cytoplasm is predominantly pink with a slight amount of blue; moderate in amount with no granules

Reference range is 5-10% in bone marrow

Diffusely Basophilic Erythrocyte

Also known as polychromatophilic erythrocyte or reticulocyte

Reported as polychromasia on a CBC (graded as a 0 to 3+)

Cell size is 7-10 micrometers

No nucleus

Cytoplasm is bluish color due to the presence of RNA
New methylene blue or brilliant cresyl blue stains the RNA and organelles to confirm these cells as reticulocytes

Reference range in peripheral blood for reticulocytes is 2.5%-6.0% in newborns and 0.5%-2.0% in adults

Erythrocyte

After the reticulocyte stage, the mature erythrocyte is formed that has the following characteristics:

Functions to transport oxygen to the tissues via hemoglobin

Survives in circulation for 120 days

Metabolizes glucose through anaerobic glycolysis

Has an average diameter of 6 to 8 micrometers

Lacks the ability to make hemoglobin

Lacks a nucleus and functional organelles

What cell is this?

Rubriblasts

What cell is this?

Prorubricyte

What cell is this?

Rubricyte

What cell is this?

Metarubricyte

What cell is this?

Diffusely Basophilic Erythrocyte

What cell is this?

Reticulocyte

What cell is this?

Erythrocyte

What cells are these?

Red arrow: Rubriblast

Blue arrow: Rubricytes

Select the nRBC

5

Select the nRBC

7

Select the nRBC

6

RBC Structure and Function

Red blood cell survival and function is dependent upon

1. 1. RBC membrane

2. 2. RBC metabolic pathways

3. 3. Hemoglobin (Hgb) structure and function

RBC Membrane

Consists of three areas

1. 1. Outer hydrophilic layer

2. - Glycolipid

3. - Glycoprotein

4. - Protein

5. 2. Central hydrophobic layer

6. - Protein

7. - Cholesterol

8. - Phospholipid

9. 3. Inner hydrophilic layer

10. - Protein

Membrane Characteristics of Erythrocytes

RBC membrane characteristics

Highly elastic: The shape of the erythrocyte constantly changes as it moves through the circulation and performs extremely complex maneuvers

The cell membrane is deformable and tolerant against mechanical stress and various pH and salt concentrations

Cell shape changes reversibly without fragmentation depending on ATP level in the cell and intracellular calcium ion concentration

The cellular membrane is composed of a protein-lattice lipid bilayer to which the membrane skeleton is attached by peripheral proteins; this network of proteins is responsible for maintaining the strength, shape, stability, and flexibility of the red blood cell to withstand the shear forces in circulation

RBC Membrane

Red blood cell membrane proteins

Integral (transmembrane): Proteins that extend from the outer surface and traverse the entire membrane to the inner cytoplasmic side of the RBC types shown in table 5.3

Peripheral: Proteins located in the cytoplasmic surface of the membrane, which is located beneath the lipid bilayer and forms the RBC cytoskeleton; involves proteins that are:

• Membrane associated

• Glycosyl phosphatidylinositol (GPI) anchored

• Cytoskeletal proteins

RBC Membrane - Peripheral Proteins

Cytoskeleton is composed of peripheral proteins that control cell shape, attachment to other cells, and maintain organization of specialized membrane domains

Types of peripheral proteins

• Spectrin (alpha and beta)

• Ankyrin (band 2.1) links the membrane skeleton to the bilayer

• Actin (band 5)

• Band 4.1 and 4.2, adducin, and p55

Together these components form a complex meshwork tethered to the RBC membrane

RBC Membrane - Glycophorin

Principle RBC glycoprotein (20% of membrane protein)

Exposed on the outer RBC membrane

Accounts for most of the membrane sialic acid, which gives RBCs their negative charge causing the RBCs to repel each other as they move through circulation

RBC Membrane - Spectrin

Important factor in RBC membrane integrity

Flexible rod-like molecule composed of 2 helical polypeptide chains (1 alpha and 1 beta)

Binds with other peripheral proteins such as actin, ankyrin, adducin, and others to form a skeletal network of microfilaments on the inner surface of the RBC membrane

Microfilaments strengthen the membrane, control the biconcave shape and deformity, and provide stability to the lipid bilayer interface

RBC Membrane - Lipids

Red blood cell membrane lipids

Three main types

1. Phospholipids

2. Glycolipids

3. Cholesterol

Red Blood Cell Membrane Lipids - Phospholipids

Choline Phospholipids: Located on the outside of the lipid bi-layer; types include:

• Phosphatidyl choline

• Sphingomyelin

Amino Phospholipids: Located exclusively on the inside or cytoplasmic side of the RBC membrane; types include:

• Phosphatidylethanolamine

• Phosphatidylserine

• Phosphatidylinositol

Red Blood Cell Membrane Lipids - Glycoproteins

Most are located in the outer half of the lipid bi-layer; they interact with glycoproteins to form many RBC antigens

Red Blood Cell Membrane Lipids - Cholesterol

Located on both sides of the lipid bi-layer inserted between the choline and amino phospholipids

Comprises 25% of the RBC membrane lipid

Continual exchange with plasma cholesterol

Accumulation of cholesterol will result in the formation of target cells and may cause membrane damage

Acanthocytes have also been associated with an excess accumulation of membrane cholesterol in certain disorders

• Liver disease

• Inherited lipid disorders such as abetalipoproteinemia and lecithin-cholesterol acyltransferase (LCAT) deficiency

RBC Deformability or Flexibility

RBC’s must be flexible as they flow throughout the circulatory system

Some biochemical changes can alter RBC flexibility and shape change such as:

1. Low concentration of fatty acids and phospholipids

2. A variety of amphoteric agents

3. Elevated intracellular calcium concentration

4. Decrease ATP levels

RBCs may be sequestered and removed by the spleen when they become aged, damaged, less flexible, or fragmented

RBC Permeability

Permeable to water and anions such as chloride and bicarbonates

Relatively impermeable to cations such as sodium and potassium

• K+ is primarily found inside the red cell (25:1 intracellular to extracellular ratio)

• Na+ is primarily found outside the red cell (1:12 intracellular to extracellular ratio)

Controls the volume of RBC and water homeostasis

Prevents osmotic hemolysis

Proteins located in the cytoplasmic surface of the membrane located beneath the lipid bilayer and forms the RBC skeleton are known as

Peripheral proteins

The peripheral protein that forms a flexible rod-like dimer responsible for the majority of the RBC membrane integrity is known as

Spectrin

Metabolic Activities of Erythrocytes

The enzymes synthesized during early cell development have to be sufficient to provide the energy needed for several processes

• Maintaining hemoglobin iron in an active ferrous (Fe 2+) state

• Driving the cation pump needed to maintain intracellular sodium ion (Na+) and potassium ion (K+) concentrations despite the presence of a concentration gradient

• Maintaining the sulfhydryl groups of globins, enzymes, and membranes in an active reduced state

• Preserving the membrane integrity

If metabolic pathways are blocked or inadequate, the life span of the erythrocyte is reduced and hemolysis results.

Defects in metabolism can include the following:

• Failure to provide sufficient reduced glutathione, which protects other elements in the cell from oxidation.

• Insufficient energy-providing coenzymes such as reduced nicotinamide-adenine dinucleotide (NADH), nicotinamide-adenine phosphate dehydrogenase (NADPH), and ATP

The two most common erythrocytic enzyme deficiencies, which involves the Embden-Meyerhof glycolytic pathway, are deficiencies of:

Glucose-6-Phosphate dehydrogenase (G6PD): responsible for converting glucose-6-phosphate (G6P) to 6-phosphogluconate (6PG)

Pyruvate kinase (PK): responsible for converting pyruvate (pyruvic acid) to lactic acid

The mature erythrocyte has no nucleus or other organelles but is capable of existing in the blood circulation for an average of 120 days.

An erythrocyte has a limited ability to me