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

Erythropoiesis and RBC maturation

Sample

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)

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.

Pathophysiology of Erythropoiesis

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 get matured in the microcirculation of the bone marrow and then are released into the circulation.
   5. In the case of increased demand, intramedullary hematopoiesis may be seen.

The various sites of erythropoiesis (Hematopoiesis):

Red blood cells hematopoiesis

Erythropoiesis is the entire process of producing RBCs in the bone marrow in response to erythropoietin.

1. Approximately 10¹² new RBCs are produced each day.
2. It takes roughly 5 days to cycle in the bone marrow.
3. In the peripheral blood, it takes 1 to 2 days.
4. One stem cell by progressive cellular division gives rise to  14 to 16 RBCs.
5. Stem cells have 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): RBCs are biconcave and contain protein, mainly hemoglobin.

1. This biconcave shape gives more surface area to combine with oxygen.
2. RBCs can change their shape to pass through the smaller capillaries.
3. RBC flexibility help to fulfill all the functions like:
   1. 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 applied stress of fluid forces, and can undergo large membrane extension without undergoing any fragmentation.

Red blood cell structure

Red blood cell structure

Red blood cell chemical structures and layers

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, will form matures RBC. This process takes place under the influence of erythropoietin.

The committed stem cells develop under the influence of burst-forming units (BFU).

Red blood cells maturation in the bone marrow and peripheral blood

Erythropoiesis and the role of erythropoietin

Differentiating points in the RBC stages morphology:

 Maturation in the peripheral blood occurs from the reticulocytes and transforms into mature red blood cells.

1. RBCs take 1 to 2 days to mature as red blood cells.
2. Maturation in the bone marrow is under the influence of a colony-forming unit (CFU).
3. The normal RBC life span is 120 days, and it can travel around 300 miles  (480 km) in circulation.

Erythropoiesis

4. Mature RBC 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
      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

RBC 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.
   

   Red blood cells functions in the human body

4. Hemoglobin of RBCs facilitates CO2 excretion.
5. RBCs can change shape, so they can very 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.
      

      Red blood cell energy metabolism

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

   Red blood cells’ source of energy

RBC life span:

1. RBCs 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

Procedure to count RBCs:

Hayme’s solution consists of :

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

Gower’s solution consists of:

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

Procedure:

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

4. 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.
5. Allow for 2 minutes to settle the cells.
6. Now count RBCs in the Neubauer chamber.
7. Use 40 X to count the RBCs.
   1. For RBCs, use the center square, which has 25 smaller squares.
8. Count the corner 4 squares and one central square.
   1. Count only the RBCs that fall on these squares’ left and top borders.
9. Repeat the count twice and divide by 2 to get the average.
10. The formula for RBCs count is:

• • Multiply factor = 10 x 200 / 0.2  = 10,000
  • Multiply RBCs count with 10,000 = RBCs million/cmm.

Neubauer chamber

Neubauer chamber for counting of RBCs

Increased RBC count is seen in:

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:

1. Anaemias.
2. Drugs that cause aplastic anemia.
3. G-6 PD deficiency.
4. Immune mechanism.
5. Malignancy like Hodgkin’s disease, 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. The 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:

• Please see more details in CBC and peripheral blood smear.

Questions and answers:

Q1: What is the difference between mature RBC and reticulocytes.

Q2: Is it possible that RBC can pass through the capillaries, and what is the reason for that function.