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Red Blood Cell (RBC):- Part 1 – Erythropoiesis and RBC maturation, RBC Counting Procedure

March 4, 2023HematologyLab Tests

Table of Contents

  • Erythropoiesis and RBC maturation
      • Sample for Red blood cell counting
      • Purpose of the test (Indications) for RBC counting
      • Erythropoiesis in early and adult life:
      • The various sites of erythropoiesis (Hematopoiesis):
      • Erythropoiesis (RBC maturation):
      • Structure of the red blood cells (RBCs):
      • Erythropoiesis and RBC maturation:
      • Maturation in the bone marrow:
      • Maturation of Red blood cells in the peripheral blood (Erythropoiesis and RBC maturation): 
      • Red Blood Cell functions:
      • Source of energy for RBC:
      • RBC life span:
      • Normal Values of red blood cells
    • Procedure to count RBCs:
        • Hayme’s solution consists of the following:
        • Gower’s solution consists of the following:
      • Procedure for RBC counting:
      • The formula for RBCs count is as follows:
      • Increased RBC count is seen in the following:
      • Decreased RBC count is seen in the following:
        • Differentiating points in the RBC stages morphology:

Erythropoiesis and RBC maturation

Sample for Red blood cell counting

  1. The blood sample is taken in EDTA.
  2. It is stable for 24 hours at 23 °C and 48 hours at 4°C.

Purpose of the test (Indications) for RBC counting

  1. This is advised for anemia or Polycythemia.
  2. It is a routine part of CBC.
  3. This is repeated in patients with repeated bleeding.

Erythropoiesis in early and adult life:

  1. In the first few weeks of intrauterine life, the yolk sac’s the primary site for erythropoiesis.
    1. The liver and spleen follow this from 6 weeks to 6 to 7 months of intrauterine life. These will continue to produce blood cells until about 2 weeks after the birth.
    2. Bone marrow also takes part and starts from 6 to 7 months of intrauterine fetal life.
    3. During childhood and adulthood, life bone marrow is the main site of erythropoiesis.
    4. The RBCs mature in the bone marrow microcirculation and then are released into circulation.
    5. In the case of increased demand, intramedullary hematopoiesis may be seen.

The various sites of erythropoiesis (Hematopoiesis):

Stages of human development Age  Site of the erythropoiesis
Fetus 0 to 2 months Yolk sac (mesoderm). (It declines by 6 weeks and ends by 2 months)
  • Fetus
  • 2 to 7 months
  1. Liver and spleen
  2. Nucleated RBCs migrate to the liver until 7 months.
  3. From 3rd to 6th months spleen also form RBCs
  • Fetus
  • 5 to 9 months
  1. Bone marrow
  2. Around the 7th month of fetal life, hematopoiesis shifts from the liver to the bone marrow.
  3. Now bone marrow is the major site for hematopoiesis
  4. Fetal marrow is filled with RBCs during hematopoiesis
  • Infants
  • Birth to 4 years
  1. Bone marrow (Practically all the bones)
  2. At birth, the liver and spleen stop the process of hematopoiesis
  3. By 4 years of age, fat cells begin to appear in the long bones
  • Adult
  • 18 to 20 years of age
Hematopoiesis is only found in the following:

  1. Vertebrae
  2. Ribs
  3. Sternum
  4. Skull
  5. Sacrum and pelvic bones
  6. The proximal end of the femur
  • Adult
  • After the age of 40 years
Bone marrow consists of an equal amount of fat and hematopoietic tissue:

  1. Sternum
  2. Ribs
  3. Pelvis
  4. Vertebrae
Erythropoiesis and RBC maturation: Red blood cells hematopoiesis

Erythropoiesis and RBC maturation: Red blood cells hematopoiesis

Erythropoiesis (RBC maturation):

  1. It is the entire process of producing RBCs in the bone marrow in response to erythropoietin.
  2. Approximately 1012 new RBCs are produced each day.
  3. It takes roughly 5 days to cycle in the bone marrow.
  4. In the peripheral blood, it takes 1 to 2 days.
  5. By progressive cellular division, one stem cell gives rise to  14 to 16 RBCs.
  6. Stem cells have a self-renewal capacity, so the bone marrow cellularity remains constant in a normal healthy state.
    1. One stem cell can produce 106 mature blood cells after 20 subdivisions.

Structure of the red blood cells (RBCs):

  1. RBCs are biconcave and contain protein, mainly hemoglobin.
  2. This biconcave shape gives more surface area to combine with oxygen.
  3. RBCs can change their shape to pass through the smaller capillaries.
  4. RBC flexibility help to fulfill all the functions:
    1. The biconcave disc can generate energy as adenosine triphosphate (ATP) by the anaerobic glycolytic (Embden-Meyerhof) pathway.
      RBC membrane by E/M shows a trilaminar structure consisting of dark-light-dark layers.
    2. These membranes indicate:
      1. Outer is a hydrophilic membrane consisting of glycolipids, glycoproteins, and protein.
      2. Central is a hydrophobic layer containing protein, cholesterol, and phospholipids.
      3. The inner layer is hydrophilic, consisting of protein.
      4. The RBC membrane is highly elastic, responds to the applied stress of fluid forces, and can undergo large membrane extension without undergoing any fragmentation.
Erythropoiesis and RBC maturation: RBC chemical structure

Erythropoiesis and RBC maturation: RBC chemical structure

Erythropoiesis and RBC maturation: Red blood cells chemical structures and layers

Erythropoiesis and RBC maturation: Red blood cell chemical structures and layers

Erythropoiesis and RBC maturation:

Maturation in the bone marrow:

  1. Bone marrow provides a suitable environment for stem cell survival, growth, and development. This consists of stromal cells and a microvascular network.
    1. Stromal cells consist of adipocytes, fibroblasts, endothelial cells, and macrophages.
  2. RBC develops from the erythroblasts in the bone marrow, which form from the stem cells.
  3. The stem cell becomes committed to stem cells under the colony-forming unit (CFU) influence.
  4. Committed stem cell becomes pronormoblast.
  5. Pronormoblast transforms into early normoblast under the influence of a burst-forming unit (BFU).
  6. Erythroblast transforms into normoblast, which will form matures RBC. This process takes place under the influence of erythropoietin.
  7. The committed stem cells develop under the influence of burst-forming units (BFU).
Erythropoiesis and RBC maturation: RBC maturation time

Erythropoiesis and RBC maturation: RBC maturation time

Erythropoiesis and RBC maturation: RBC effect of erythropoietin

Erythropoiesis and RBC maturation: RBC effect of erythropoietin

Differentiating points in the RBC stages morphology:

Features Pronormoblast Normoblast Reticulocyte Mature RBC
Cell size µm 14 to 19 12 to 17 7 to 10 7 to 8
Nuclear shape round round absent absent
Nuclear chromatin reddish-blue blue purple absent absent
Nucleoli 0 to 2 absent absent absent
Cytoplasm dark or royal blue pink, moderate clear, gray-blue pink

Maturation of Red blood cells in the peripheral blood (Erythropoiesis and RBC maturation): 

  1. It occurs from the reticulocytes and transforms into mature red blood cells.
  2. RBCs take 1 to 2 days to mature as red blood cells.
  3. Bone marrow maturation is influenced by a colony-forming unit (CFU).
  4. The normal RBC life span is 120 days, and it can travel around 300 miles  (480 km) in circulation.
Erythropoiesis and RBC maturation: Erythropoiesis process

Erythropoiesis and RBC maturation: Erythropoiesis process

  1. Mature RBCs can be differentiated from the normoblast and reticulocytes.
    1. The normoblastic cells have a prominent nucleus, while reticulocytes have remnants of nuclear chromatin (RNA).
Characteristic features Red blood cell (RBC) Reticulocyte Normoblast
Morphology Normal RBC Reticulocytes Normoblast
Presence of nucleus (DNA) Absent Absent Present
Presence of RNA in the cytoplasm Absent Present Present
Location Peripheral blood Peripheral blood + Bone marrow  Bone marrow
Presence in the peripheral blood Present Present Absent
Proliferation Absent Absent Present
Heme synthesis Absent Present Present
Presence of mitochondria Absent Present Present
Protein synthesis Absent Present Present
Lipid synthesis Absent Present Present
Embden-Meyerhof pathway Present Present Present
Pentose phosphate pathways Present Present Present

Red Blood Cell functions:

  1. The RBCs’ primary function is to carry oxygen from the lungs to other body tissue and deliver CO2 from the tissue to the lung.
  2. After oxygenation in the lung, RBC carries  O2 back to body tissue.
  3. RBCs are biconcave, giving Hb more surface area to combine with O2.
Erythropoiesis and RBC maturation: RBC role in oxygen transport

Erythropoiesis and RBC maturation: RBC’s role in oxygen transport

  1. Hemoglobin of RBCs facilitates CO2 excretion.
  2. RBCs can change shape, so they can easily pass through the small capillaries.

Source of energy for RBC:

  1. RBC utilizes glucose, which generates 2 ATP molecules that generate energy to maintain:
    1. Hb function.
    2. RBC membrane.
    3. RBC  volume.
    4. RBC shape
    5. RBC flexibility.
    6. Adequate amounts of reduced pyridine nucleotide.
Erythropoiesis and RBC maturation: Glucose as a source of energy for RBCs

Erythropoiesis and RBC maturation: Glucose as a source of energy for RBCs

  1. Glucose is metabolized in the RBC, and it needs a Glucose-6-phosphate dehydrogenase enzyme to convert glucose to Fructose-6-phosphate.
  2. RBCs generate energy almost exclusively through the anaerobic breakdown of glucose.
  3. The adult RBCs possess little ability to metabolize fatty acids and amino acids.
  4. Mature RBCs do not possess mitochondria for oxidative metabolism.
Erythropoiesis and RBC maturation: RBCs source of energy

Erythropoiesis and RBC maturation: RBCs source of energy

RBC life span:

  1. RBC life in the peripheral blood is around 120 days.
  2. The aged RBCs are extracted from the blood by the spleen.
  3. Abnormal RBCs have a shorter lifespan.
  4. Hypersplenism may destroy the RBCs and remove them from circulation.
  5. There are approximately 500 RBCs for one WBC.

Normal Values of red blood cells

Source 1

  • Cord blood = 3.9 to 5.5 million/cmm
  • Adult = 18 to 44 years :
    • Male = 4.7 to 6.1 million/cmm.
    • Female = 3.8 to 5.4 million/cmm
  • 45 to 64 years :
    • Male = 4.2 to 5.6 million/cmm.
    • Female = 3.8 to 5.0 million/cmm
  • 65 to 74 years :
    • Male = 3.8 to 5.8 million/cmm.
    • Female = 3.8 to 5.2 million/cmm

Source 4

Age  Normal value
Birth to 2 weeks 4.1 to 6.1 x 106/cmm
2 to 8 weeks 4.0 to 6.0 x 106/cmm
2 to 6 months 3.8 to 5.6 x 106/cmm
6 months to 0n3 year 3.8 to 5.2 x 106/cmm
1 to 6 years 3.9 to 5.3 x 106/cmm
6 to 16 years 4.0 to 5.2 x 106/cmm
16 to 18 years 4.2 to 5.4 x 106/cmm
>18 years male 4.5 to 5.5 x 106/cmm
>18 years female 4.0 to 5.0 x 106/cmm
Men 4.2 to 5.4 x 106/cmm
Women 3.6 to 5.0 x 106/cmm

Procedure to count RBCs:

Hayme’s solution consists of the following:

  1. Na Cl = 1 G (Isotonic solution).
  2. Na2SO4 = 5 grams. It will prevent rouleux formation.
  3. HgCl2 = 0.5 G acts as an antiseptic.
  4. D. H2O = 200 mL

Gower’s solution consists of the following:

  • Na Cl for an isotonic solution.
  • Na2SO4 = 12.5 G
  • Glacial acetic acid = 33.3 G
  • D.H2O = 200 mL

Procedure for RBC counting:

  1. RBCs counting solution is Hayem’s or Gowers isotonic saline.
  2. Make a dilution of 1:200 with a diluting solution. Fill the red bulb pipette up to 0.5 marks with the blood.
  3. Draw the solution to mark 101 of the RBC pipette.
Red blood cell (RBC) pipette

Red blood cell (RBC) pipette

  1. Mix the blood thoroughly in the pipette.
    1. Discard the first few drops (4 to 5 ) and fill the Neubauer chamber.
    2. Make sure that the chamber is free of air bubbles.
    3. The distribution of the cells should be uniform over the ruled area.
  2. Allow for 2 minutes to settle the cells.
  3. Now count RBCs in the Neubauer chamber.
  4. Use 40 X to count the RBCs.
    1. For RBCs, use the center square, which has 25 smaller squares.
  5. Count the corner 4 squares and one central square.
    1. Count only the RBCs that fall on these squares’ left and top borders.
  6. Repeat the count twice and divide by 2 to get the average.

The formula for RBCs count is as follows:

  • Multiply factor = 10 x 200 / 0.2  = 10,000
  • Multiply RBC count by 10,000 = RBCs million/cmm.
Neubauer chamber

Neubauer chamber

Neubauer chamber for counting of RBCs

Neubauer chamber for counting of RBCs

Increased RBC count is seen in the following:

  1. Primary Erythrocytosis.
    1. Polycythemia.
    2. Erythremia (Erythrocytosis).
  2. Secondary Erythrocytosis.
    1. Vigorous exercise.
    2. Hemoconcentration.
    3. High Altitude.
    4. Chronic obstructive pulmonary disease (COPD).
    5. Severe dehydration.
    6. Thalassemia trait.
    7. Hemoglobinopathies.
    8. Congenital heart disease.
    9. Extra-renal tumors.
    10. Tobacco use.

Decreased RBC count is seen in the following:

  1. Anaemias.
  2. Drugs that cause aplastic anemia.
  3. G-6 PD deficiency.
  4. Immune mechanism.
  5. Malignancy like Hodgkin’s disease and lymphomas.
  6. Acute and chronic hemorrhage.
  7. Autoimmune diseases like SLE and rheumatoid arthritis.
  8. A chronic infection like subacute endocarditis.
  9. Cirrhosis.
  10. Dietary deficiency of iron and vit B12.
  11. Pregnancy.
  12. Marrow failure, e.g., Bone Marrow fibrosis, leukemia infiltration, chemotherapy, and antiepileptic drugs.
  13. Drugs leading to bone marrow failures like quinidine, chloramphenicol, and hydantoin.
  14. Hemolysis is seen in spherocytosis, G6PD deficiency, and splenomegaly.
  15. Genetic abnormality is seen in thalassemia and sickle cell anemia.
  16. Hemorrhage, e.g., in GI tract or trauma.
  17. Chronic illness due to infections or malignancies.
  18. Organ failure is seen in renal diseases.

Differentiating points in the RBC stages morphology:

Features Pronormoblast Normoblast Reticulocyte Mature RBC
Cell size µm 14 to 19 12 to 17 7 to 10 7 to 8
Nuclear shape round round absent absent
Nuclear chromatin reddish-blue blue purple absent absent
Nucleoli 0 to 2 absent absent absent
Cytoplasm dark or royal blue pink, moderate clear, gray-blue pink
  • Please see more details in CBC and peripheral blood smear.

Questions and answers:

Question 1: What is the difference between mature RBCs and reticulocytes?
Show answer
RBCs lakes nuclear reticulin while there are remnants of nuclear reticulin in the reticulocytes.
Question 2: Is it possible that RBC can pass through the capillaries and what is the reason for this function?
Show answer
RBCs are biconcave and these can pass through the capillaries, and RBC membrane is highly elastic.

Possible References Used
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