07 August 2017

Lecture #3: The Red Blood Cells


ERYTHROPOIESIS


It is believed that the pluripotential hematopoietic stem cell is induced by certain microenvironmental influences to become the committed erythroid progenitor cell which is sensitive to erythropoeitic stimulation. In response to a hormone, erythropoietin, the committed progenitor cells in the marrow undergoes mitosis and one or both daughter cells enter the erythroid maturation sequence.

The committed progenitor cell gives rise by mitosis to immature erythroblasts which are capable of further division and at the same time mature into semi–mature erythroblasts. These in turn develop into mature erythroblast which on losing their nuclear substance by dissolution from reticulocytes. The maturation sequence from rubriblast to reticulocyte in the bone marrow takes approximately seven days. The reticulocyte then remains another three and a half days or go into the bone marrow before issuing into the bloodstream where it remains recognizable as a reticulocyte for one or more day before discharging its oxygen–transporting function as a normal erythrocyte.


ERYTHRON


The term “erythron” has been applied to the single functional entity composed of red cells and their precursors. This includes the normoblasts at all stage of maturation, the reticulocytes as well as the erythroid–committed stem cells and the circulating erythrocytes. The interstitial tissue of the erythron is represented by the plasma and the fat and reticulum of the bone marrow.


Nutritional requirements for red cell production

a. Proteins and amino acids
b. Vitamins – Vitamin B12, Folic acid, Vitamin B6, riboflavin, panthotenic acid, nicotinic acid, ascorbic acid.
c. Minerals


Iron metabolism


The metabolism of iron is dominated by its role in hemoglobin synthesis. When synthesis is complete, the iron now in the form of hemoglobin in mature red cells is delivered to the circulation. At the end of the 120 days life span, the red cells are engulfed by macrophages of the reticuloendothelial system (RES) where the iron is extracted from the hemoglobin. Some of this iron may remain stored in the RES as ferritin of hemosiderin, but most is delivered to the plasma where it becomes bound to transferrin, completing the iron cycle.


Control of erythropoiesis


Alterations in the concentration of hemoglobin in the blood lead to charges in tissue oxygen tension within the kidney. In response to hypoxia, the kidney secretes a factor that interacts with plasma substrate to produce a hormone, erythropoietin. This hormone induces primitive marrow cells to differentiate into pronormoblasts, thereby bringing about expansion of the erythroid marrow and an increase in red cell production. This in turn leads to an increase in the size of the erythron and increase in tissue oxygen levels.


Synthesis of Hemoglobin – Heme (iron) plus Globin (protein)


a. Heme synthesis occurs in most red cells of the body, except the mature erythrocyte, but most abundantly in the erythroid precursor.

b. Globin synthesis occurs in the cytoplasm of the erythroblast and reticulocyte.

Hemoglobinization occurs while the erythrocyte is developing in the bone marrow and still possesses a nucleus. It usually begins during the latter half of the polychromatophilic normoblast stage. Hemoglobinization is completed while the cell still possesses a nucleus. However, it can continue in the reticulocyte stage, although the rate of synthesis is greatly reduced because there is no nucleus.


Structure of erythrocytes


The mature erythrocytes are 6 to 8 micrometers in diameter. They have a thickness of 1.5 to 2.5 micrometers (average of 2), a corpuscular volume of 75 to 95 femtoliters (average is 87) and a surface area of 130 to 150 square micrometers (average is 135).

Under the microscope, unstained red cells have light greenish yellow color. Red cells stain a buff of reddish color. Their shape is that of a biconcave disc, thus called discocyte causing the cells to appear lighter in the center than in the periphery. Some of the cells may have cup or spherical shapes. The shape of erythrocytes can change tremendously as they pass through the capillaries. Actually, red blood cells look like a “bag” that can be deformed into almost any shape.


Composition of erythrocytes


1. 60% water and 40% solids

2. Hemoglobin – iron bearing protein which serves as the most important agent in the erythrocyte

3. Red cell membrane which is composed of:

a. Stroma – the innermost structure of the erythrocyte which is composed primarily of protein and lipids. The hemoglobin attaches itself in an interlacing manner to the stroma.

b. Membrane – composed of lipoproteins. This membrane is extremely thin and pliable. It is a dynamic semi–permeable one, retaining potassium in high concentration within the cell and excluding sodium, while allowing hydrogen, chloride and bicarbonate ions to pass freely into or out of the cell in proportion to the ionic gradient.

4. Red cell enzymes

a.      Carbonic anhydrase
b.      Methemoglobin reductase
c.       Catalase
d.      Glucose–6–phosphate dehydrogenase
e.      Pyruvate kinase
f.        Adenosine deaminase
g.      Aldolase
h.      Lactic dehydrogenase
i.        Glutathione reductase
j.        Nucleoside phosphorylase
k.       Acetylcholinesterase


Functions of erythrocytes


1. To mediate the exchange of respiratory gases, oxygen and carbon dioxide between the lungs and tissues.

2. To control the blood pH to assist in the maintenance of acid–base equilibrium


Erythrocyte destruction


The erythrocyte gradually undergoes metabolic changes over the course of its 120 day life span, at which time the less viable senescent cell is removed from the circulation. Certain glycolytic enzymes diminish in activity as the cell ages. Older red cells have a smaller surface area and in increased MCHC compared with younger cells. Changes in the cell surface may render the cell more liable to phagocytosis. The exact mechanism by which senescent erythrocytes are recognized and removed by the reticuloendothelial system is unknown. The process may be phagocytosis of whole erythrocytes or fragmenting senescent cells.


Mechanism of red cell destruction


1. Fragmentation – loss of a portion of the erythrocytes membrane, accompanied by loss of cellular contents, including hemoglobin

2. Osmotic lysis – the passing of water into the red cell on such a scale as to ultimately burst it (hemolysis)

3. Electrophagocytosis – ingestion of whole red cells by circulating monocytes or neutrophils or by fixed macrophages of the reticuloendothelial system (RES)

4. Complement induced cytolysis – complement has the ability to attach to the cells and induce lysis. This is the usual event that triggers cellular fixation of complement, that is the reaction between the cellular antigen and a humoral factor.

5. Hemoglobin denaturation – when erythrocytes are exposed to oxidant stress and the mechanism to protect the cell from such damage fails to work, denatured hemoglobin precipitates forming inclusion bodies known as Heinz bodies.


Site of erythrocyte destruction


1. Intravascular hemolysis – lysis of erythrocytes occurs within the circulation, through the classic pathway. It is the usual outcome of sensitization of erythrocytes with complement

2. Extravascular hemolysis – lysis of erythrocyte outside the circulation but in the reticuloendothelial cells of the liver and spleen. This usually happens through phagocytosis


Intra and extracellular hemolysis


1. Intracellular or intracorpuscular hemolysis – lysis of the red cells is due to intracorpuscular defects like abnormalities in RBC membrane, deficiency in enzymes, abnormalities in synthesis of hemoglobin.

2. Extracellular or extracorpuscular hemolysis – lysis of the red cells is due to extracorpuscular defects or factors like infectious agents, chemicals, drugs, antibodies, venous factors, physical agents, etc.


Erythrocyte count


The erythrocytes occupy the largest function of the formed elements of the blood. As the body functions normally, the blood count remains stable, but physiologic as well as pathologic conditions alter the red blood cell count.

Adult male                  4,500,000 – 6,500,000                        4.5 – 6.5 x 1012/L
Adult female               4,000,000 – 5,000,000                        4 – 5 x 1012/L
Infants                         4,000,000 – 6,000,000                        4 – 6 x 1012/L
Children                      4,000,000 – 5,700,000                        4 – 5.7 x 1012/L


RED CELL ABNORMALITIES


In diseases, erythrocytes vary in their hemoglobin content, size, shape, staining properties and structure

A. Variation in hemoglobin content

Irregularities in hemoglobin distribution are usually due to the shape of the cell and to degenerative changes or abnormalities of cell formation particularly hemoglobin synthesis

1. Normochromic cell – refers to erythrocyte with normal amount of hemoglobin

2. Hypochromic  cell – refers to erythrocyte wherein the central light area of the cell is larger and paler than normal

a. Hypochromia in erythrocytes with normal size indicates that there is less than the normal amount of hemoglobin present in the cell.

b. Hypochromia in erythrocytes with larger than normal size (macrocytes) indicates that a normal amount of hemoglobin may be present due to the increased size of the cells, a hypochromic effect is still produced.

Hypochromia is found in

Iron deficiency anemia                                   In parasitism
Hemorrhages                                      In case of malignancy
Thalassemia                                        Sideroblastic anemia

3. Hyperchromic cell – refers to erythrocyte which has an increased hemoglobin content and wherein the central light area is smaller than normal or non–existent.

In spherocytosis, the cells are hyperchromic, though the hemoglobin content is normal, the hemoglobin concentration is increased due to reduced surface / volume ratio.

Hyperchromia is seen in:

Megaloblastic anemia                              Severe diarrhea
Hereditary spherocytosis                          Heart disease

4. Anisochromia – a condition wherein both hypochromic and normochromic cells are present in the same blood film. It is sometimes called dimorphic anemia. This is found in:

Sideroblastic anemia
Iron deficiency anemia after iron therapy

B. Variation in staining property

1. Polychromatophilia or polychromasia or diffuse basophilia

This is a condition wherein the red cells are stained with various shades of blue with tinges of pink. This is due to the combination of the affinity of hemoglobin to acid stain and the affinity of ribonucleic acid (RNA) to the basic dye. This is a characteristic of young or immature red cells with residual RNA; cells are larger than normal and correspond to reticulocytes. This condition is found in:

Reticulocytosis                  Acute blood loss
Hemolysis                         Pernicious anemia and other anemia
Punctuate basophilia or basophilic stippling of RBC

This is a special form of polychromasia in which basophilic granules usually isolated either fine or sometimes coarse appear in the cytoplasm of erythrocytes. This is due to vacuolar degeneration of the polychromatic substance of the cytoplasm; the stippling probably represents aggregated RNA. This is found in:

Toxic anemia                                            Lead poisoning
Hemolytic anemia                                    Thalassemia
Leukemia                                      Other congenital forms of anemia

2. Hypochromasia – a condition wherein the red cells stain usually palely. There are two possible causes: a lowered hemoglobin concentration and abnormal thinness of the red cells. This is found in:
           
Iron deficiency anemia                 Sideroblastic anemia
            Thalassemia

3. Hyperchromasia – a condition wherein the red cells are stained deeply due to abnormal thickness of the cells. This is found in:

            Macrocytosis                           Megaloblastic anemia
            Spherocytosis

4. Anisochromia – a condition wherein a proportion only of the red cells stains palely, and can be found in:

            Iron deficiency anemia responding to iron therapy
            Sideroblastic anemia

5. Anulocyte or “ring” cells – these are thin cells with low hemoglobin content and are represented by thin stain ring surrounding a large central space.

C. Variation in size

1. Anisocytosis – a condition wherein the red cells vary in size, both macrocytes and microcytes coexist in the same smear

2. Normocyte – 6 – 8 micrometers in diameter; this is an erythrocyte with a normal size

3. Macrocyte – cell with 10 – 12 micrometers in diameter; characteristic of young red cell of skipped generation with early loss of nucleus. The cell is well filled with hemoglobin.

This is found in: pernicious anemia, aplastic anemia, cirrhosis of the liver

4. Microcyte – cell with less than 5 micrometers in diameter; this is small and round red cell, and is formed as such, or results from fragmentation. Microcytosis is found in:

            Iron deficiency anemia           Hemolytic anemia
            Thalassemia                            Sideroblastic anemia

5. Megalocyte – large oval–shaped red cell with over 12 micrometers in diameter with impaired DNA synthesis.

It is found in:

Megaloblastic anemias like pernicious anemia or Vitamin B12 deficiency anemia,
Folic acid deficiency anemia

D.     Variation in shape



1. Poikilocytosis – condition wherein the red cells exhibit variation in shape

2. Discocyte – normal cell with biconcave disc shape

3. Target cell – also known as leptocyte or Mexican hat cell or cell with a Bull’s eye appearance
                         
– this is an erythrocyte with a distinct peripheral and central zone of
            hemoglobin and annular area or pallor in between.

            – target cells are thought to result from cells having a surface which is
                        disproportionately large compared with their volume. This
                        leptocytosis is found in thalassemia, after splenectomy, chronic liver
            disease, iron deficiency anemia, certain hemoglobinopathies

4.      Elliptocyte – also known as ovalocyte. Found in:
           
Healthy persons
            Megaloblastic anemia
            Hypochromic anemia

5.      Sickle cell – crescent shaped cell due to abnormal aggregation of HbS which gives
                        a tendency for the cell to assume a sickle shape in deprivation of
                        oxygen. Also known as Drepanocyte or meniscocyte. Found in sickle
                        cell anemia and sickle cell trait which is common among Negroes.

6.      Spherocyte – a special form of microcyte approximately 6 micrometer in
                        diameter which is more spheroidal than normal red cell. This results
                        from genetic defects of the red cell membrane. It is found in
                        hereditary hemolytic anemia

7.      Schistocytes – fragmented or greatly distorted red cells which are helmet or
                        triangular or irregular in shape; red blood cells lose fragments after
                        impact with fibrin strands, walls of diseased blood vessel and artificial
                        surfaces in the circulation

8.      “Tear drop” cell – red blood cell which has a shape resembling a drop, a type of
                                    distorted or Fragmented RBC; found in myelofibrosis and
                                    thalassemia

9.      Fragmentocytes – fragmented red cells which are sometimes called “eggshells”;
                                    found in diseases involving intravascular clotting.

10.  Stomatocytes – red cells in which the central biconcave area appears slit–like in
                                    dried films. In Wet preparations, the cells are cup–shaped;
                                    particularly present in blood smears of Australian or
                                    Mediterranean origin; found in alcoholic cirrhosis and
                                    other liver diseases, hereditary hemolytic anemia

11.  Pyknocyte – distorted and contracted red blood cell similar to echinocyte; found
                                    in  infantile pinocytosis.

12.  Spiculed cells

a. Crenated cells – red cells which develop many or numerous projections from their surface; crenation can result from many causes, e.g., by washing red cells free from plasma and suspending them in NSS between glass surfaces; crenation may also result from suspension of cells in hypertonic solution; can be found in uremia.

b. Echinocytes – also called “sea–urchin” cells or burr cells; small cells or cell fragments bearing 10 to 30 spines or spicules evenly distributed over the surface of the cells; echinocytes may be caused by accumulation of fatty acid or lyso– lecithin on RBC surface; found in uremia, hemolytic anemia, post splenectomy, pyruvate kinase deficiency.

c. Acanthocytes – also known as spur cells; red blood cells with spiny projections of various lengths and irregular spacing; have 5 to 10 spicules; abnormality is due to increased ratio of cholesterol/lecithin on the RBC membrane; found in liver disease, hemolytic anemia, post splenectomy, pyruvate kinase deficiency.

E. Variation due to presence of erythrocytic inclusion bodies

1. Malarial stippling – fine granular appearance of erythrocytes that harbor malarial parasites

            Schuffner’s granules (P.vivax)
            Maurer’s dots (P. falciparum)
            Zieman’s dots (P. malariae)

2. Punctuate basophilia – refers variation in staining

3. Siderocyte – mature red cells with iron particles in the ferric form that give a positive reaction to Perl’s reaction or Prussian blue reaction; indicative of faulty iron utilization in the synthesis of hemoglobin; found in hemolytic anemia and chemical poisoning

4. Sideroblast – nucleated red cell containing siderotic granules; sometimes the granules may be arranged around the nucleus in the form of a ring and the cells are then called “ringed sideroblast”

5. Semi–lunar bodies – also known as achromocyte or crescent bodies; pale bluish–pink non–granular structures which are half-moon shaped; these bodies are degenerative remains or smudges of the RBC stroma; maybe found in malaria or hemolytic anemia

6. Howell–Jolly bodies – single or double bud bodies of varying sizes; these bodies are nuclear remnants containing DNA; these are indicative of rapid blood regeneration; maybe found in megaloblastic anemia, hemolytic anemia, leukemia, after splenectomy

7. Magraliano body – vacuole–like or elliptical body found in the center or periphery of the cell

8. Cabot rings – looped or figure of 8 shape or ring with red–purple color; ring is composed of fine granules arranged in a linear pattern; represent particles of DNA probably attached to endoplasmic reticulum; indicative of defective of regenerative activity

9. Heinz or Ehrlich bodies – intracorpuscular aggregates of denatured hemoglobin (especially globin residue); small round inclusions with intense blue color located close to the cell membrane or eve outside it; need supravital staining with methylene blue, brilliant cresyl blue or crystal violet; indicative of disturbed hemoglobin synthesis and breakdown

10.  Pappenheimer bodies – basophilic iron containing granules found in red cells stained with a Romanowsky dye, e.g. Wright’s stain; indicative of abnormal utilization of iron in the synthesis of hemoglobin

11.  Hemoglobin H inclusions – caused by instability of HbH

12.  Hemoglobin Zurich inclusions – found in drug induced conditions; infections

F. Miscellaneous variation

1. Rouleaux formation – alignment of red cells one on top of the other forming an arrangement resembling stacks of coins.

2. Crenated erythrocytes – cells with puckered outer edges found in blood smears which dry slowly

3. Partially hemolyzed RBC – slightly colored and malformed RBC caused by moisture on the slide prior to smear making






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