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

Erythropoiesis (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

1. Erythropoiesis in early and adult life:
2. In the first few weeks of intrauterine life, the primary site for erythropoiesis is the yolk sac.
   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 adult, 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):

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)
	2 to 7 months	Liver and spleen Nucleated RBCs migrate to the liver until 7 months. From 3rd to 6th months spleen also form RBCs
	5 to 9 months	Bone marrow Around the 7th month of fetal life, hematopoiesis shift from the liver to the bone marrow Now bone marrow is the major site for hematopoiesis Fetal marrow is filled with RBCs during hematopoiesis
Infants	Birth to 4 years	Bone marrow (Practically all the bones) At birth, the liver and spleen stop the process of hematopoiesis 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: Vertebrae Ribs Sternum Skull Sacrum and pelvic bones 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: Sternum Ribs Pelvis Vertebrae

1. 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 cells by progressive cellular division give 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.
2. 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 the 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 cells chemical structures and layers

3. 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, fibroblast, endothelial cells, and macrophages.
   2. RBC develops from the erythroblasts in the bone marrow, which forms 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.
   7. 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 role 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

       

4. 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.

5. 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

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

      Red blood cells functions in the human body

6. 3. Hemoglobin of RBCs facilitates CO2 excretion.
   4. RBCs can change shape, so they can very easily pass through the small capillaries.
7. 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

1. 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

9. RBC life span:
   9. RBCs life in the peripheral blood is around 120 days.
   10. The aged RBCs are extracted from the blood by the spleen.
   11. Abnormal RBCs have a shorter lifespan.
   12. Hypersplenism may destroy the RBCs and remove them from circulation.
   13. 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 :

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.
3. Draw the solution to mark 101 of the RBC pipette.
   

   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.

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:

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.

---