Red Blood Cell (RBC) – Part 4 – Erythropoiesis, Red blood cell Count and Counting Procedure
- The blood sample is taken in EDTA.
- It is stable for 24 hours at 23 °C and 48 hours at 4°C.
Purpose of the test (Indications)
- This is an indicator of anemia or Polycythemia.
- It is a routine part of CBC.
- This is repeated in patients with repeated bleeding.
- RBC count is closely related to hemoglobin and hematocrit.
- Erythropoiesis is the entire process by which the RBCs are produced in the bone marrow, in response to erythropoietin.
- It takes roughly 5 days cycle in the bone marrow.
- In the peripheral blood, it takes 1 to 2 days.
- One stem cells by progressive cellular division give rise to 14 to 16 RBCs.
- RBCs are biconcave and contain protein mainly hemoglobin.
- Maturation in the bone marrow:
- RBC develops from the erythroblasts in the bone marrow, which forms from the stem cells.
- The stem cell becomes committed to stem cells under the influence of the colony-forming unit (CFU).
- Committed stem cell becomes pronormoblast.
- Pronormoblast transforms into early normoblast under the influence of burst-forming unit (BFU).
- Erythroblast transforms into normoblast, will form matures RBC. This process takes place under the influence of erythropoietin.
- Maturation in the peripheral blood takes place from the reticulocytes and transform into mature red blood cells.
- The function of RBC is to carry oxygen from the lungs to other body tissue delivers CO2 from the tissue to the lung.
- After oxygenation in the lung, RBC carries O2 back to body tissue.
- RBCs are biconcave which gives more surface area for Hb to combine with O2.
- RBCs can change the shape so they can very easily pass through the small capillaries.
- Source of energy for RBC:
- RBC utilizes glucose which generates 2 ATP molecule which generates energy to maintain the RBC membrane, volume, shape, and its flexibility.
- Glucose is metabolized in the RBC and it needs Glucose-6-phosphate dehydrogenase enzyme for the conversion of glucose to Fructose-6-phosphate.
- RBC life span:
- RBCs life in the peripheral blood is around 120 days.
- The aged RBCs are extracted from the blood by the spleen.
- Abnormal RBCs have a shorter lifespan.
- Hypersplenism may destroy the RBCs and remove them from the circulation.
- There are approximately 500 RBCs for one WBC.
- Low RBCs count may be due to the following causes:
- Marrow failure e.g. Bone Marrow fibrosis, leukemia infiltration, chemotherapy, and antiepileptic drugs.
- Drugs leading to bone marrow failures like quinidine, chloramphenicol, and hydantoin.
- Hemolysis is seen in spherocytosis, G6PD deficiency, and splenomegaly.
- Dietary deficiency of iron and vit.B12.
- The genetic abnormality is seen in thalassemia and sickle cell anemia.
- Haemorrhage e.g. in GI tract or trauma.
- Chronic illness due to infections or malignancies.
- Organ failure as seen in renal diseases.
- 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
Procedure to count RBCs:
Hayme’s solution consists of :
- Na Cl = 1 G (Isotonic solution).
- Na2SO4 = 5 gram. It will prevent rouleux formation.
- HgCl2 = 0.5 G acts as antiseptic.
- 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
- RBCs counting solution is Hayem’s or Gowers isotonic saline.
- Make a dilution of 1:200 with a diluting solution. Fill the red bulb pipette up to 0.5 marks.
- Draw the solution to mark 101 of the RBC pipette.
- Mix the blood thoroughly in the pipette.
- Discard the first few drops (4 to 5 ) and then fill the Neubauer chamber.
- Make sure that the chamber is free of air bubbles.
- Distribution of the cells should be uniform over the ruled area.
- Allow for 2 minutes to settle the cells.
- Now count RBCs in the Neubauer chamber.
- Use 40 X to count the RBCs.
- For RBCs use the center square which has 25 smaller squares.
- Count the corner 4 squares and one central square.
- Count only the RBCs which fall on the left and top border of these squares.
- Repeat the count twice and divide by 2 to get the average.
- 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:
- Primary Erythrocytosis.
- Erythremia (Erythrocytosis).
- Secondary Erythrocytosis.
- vigorous exercise.
- High Altitude.
- Chronic obstructive pulmonary disease (COPD).
- Severe dehydration.
- Thalassemia trait.
- Congenital heart disease.
- Extra-renal tumors.
- Tobacco use.
Decreased RBC count is seen in:
- Drugs that cause aplastic anemia.
- G-6 PD deficiency.
- Immune mechanism.
- Malignancy like Hodgkin’s disease, lymphomas.
- Acute and chronic hemorrhage.
- Autoimmune diseases like SLE, and rheumatoid arthritis.
- A chronic infection like subacute endocarditis.
- Myeloproliferative disorders like leukemia and Myeloma.
- Dietary deficiency of iron, and vit B12.
Differentiating points in the RBC stages morphology:
|Cell size µm||14 to 19||12 to 17||7 to 10||7 to 8|
|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.