MLAB 1315- HematologyKeri Brophy-Martinez

Chapter 5: The Erythrocyte

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

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

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

Stem Cell

CFU-GEMM

BFU-E

CFU-E

Mature RBC

EPO

Maturation Sequence of Erythrocytes Rubriblast (Pronormoblast)

Size = 14-20 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

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

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

Maturation Sequence of Erythrocytes Metarubricyte -

Nucleated RBC (orthochromic normoblast) Size: 8-10 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

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

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.

Maturation Sequence of Erythrocytes Mature erythrocyte

Size = 7-8µm Volume = 80-100 fL Cytoplasm

Pink/red Biconcave shape

Nucleus - none Present in circulation

for about 120 days

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

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

Red Blood Cell Metabolism 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.)

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

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?

Checkpoint Embden-Meyerhof :90-95% Rapoport-Leubering shunt: oxygen affinity Hexose-Monophosphate shunt/

Methemoglobin Reductase pathway: iron

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

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.

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

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