Erythropoiesis, RBC counting Procedure

What sample is needed 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.

What are the 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.

How will you discuss Erythropoiesis in early and adult life?

1. In the first few weeks of intrauterine life, the yolk sac is 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. Bone marrow is the main site of erythropoiesis during childhood and adulthood.
   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.

What are the various sites of erythropoiesis (Hematopoiesis)?

How will you discuss Erythropoiesis (RBC maturation)?

1. It is the 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.

What is the 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 helps to fulfill all the functions:
5. The biconcave disc can generate energy such as adenosine triphosphate (ATP) through the anaerobic glycolytic (Embden-Meyerhof) pathway.
6. RBC membrane by E/M shows a trilaminar structure consisting of dark-light-dark layers.
7. 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: Red blood cell chemical structures and layers

What is Erythropoiesis and RBC maturation?

Maturation in the bone marrow:

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

Differentiating points in the RBC stages morphology:

How will red blood cells mature 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 life span of an RBC is 120 days, and it can travel around 300 miles (480 km) in circulation.

5. 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
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

What are the functions of Red Blood Cells?

1. The RBCs’ primary function is to carry oxygen from the lungs to other body tissue and deliver CO2 from the tissue to the lungs.
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.

4. Hemoglobin of RBCs facilitates CO2 excretion.
5. RBCs can change shape and easily pass through the small capillaries.

What are the Sources 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.

2. Glucose is metabolized in the RBC, and a Glucose-6-phosphate dehydrogenase enzyme is needed to convert glucose to Fructose-6-phosphate.
3. RBCs 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.

What is the life span of red blood cells (RBC)?

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.

What are the 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

What is the Procedure for counting 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 grams
• Glacial acetic acid = 33.3 G
• D.H2O = 200 mL

What is the Procedure for RBC counting?

1. RBCs counting solution is Hayem’s or Gowers isotonic saline.
2. Make a 1:200 dilution 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.
5. Make sure that the chamber is free of air bubbles.
6. The distribution of the cells should be uniform over the ruled area.
7. Allow for 2 minutes to settle the cells.
8. Now count RBCs in the Neubauer chamber.
9. Use under 40x to count the RBCs.
   1. For RBCs, use the center square, which has 25 smaller squares.
10. Count the corner 4 squares and one central square.
11. Count only the RBCs on these squares’ left and top borders.
12. Repeat the count twice and divide by 2 to get the average.

What is the formula for RBC counting?

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

Neubauer chamber

Neubauer chamber for counting of RBCs

What are the causes of Increased RBC count?

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.

What are the causes of decreased RBC count?

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.

What are the differentiating points in the RBC stages?

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

Questions and answers: