Complete blood count (CBC):- Part 2 – Red Blood Cells Morphology, Functions, Interpretations and Hemoglobin Formation

Complete blood count (CBC)

Sample for CBC

1. Takes blood in the EDTA.
2. Direct blood smears for the appreciation of the RBC morphology.

Indications

1. To diagnose the various types of anemia.
2. Any other abnormality of white blood cells and platelets.

Pathophysiology of the red blood cells:

Red blood cell metabolism is important for its survival and function.

1. Red blood cell metabolism is dependant upon:
   1. Red blood cell membrane:
      1. The RBC membrane consists of proteins and phospholipids.
      2. It can change the shape without any fragmentation or damage.
      3. The RBC membrane consists of 50% proteins, 20% phospholipids,  20% cholesterol, and 10% carbohydrates.
      4. Carbohydrates are only in the outer layer.
         

         Red blood cell membrane structure and functions

2. Hemoglobin formation, structure, and function.
   1. Normal hemoglobin formation is dependant upon:
      1. Adequate iron supply and delivery.
      2. Adequate formation of the precursor of the heme, protoporphyrins.
      3. Adequate synthesis of globin.
   2. Hemoglobin (Hb) is a conjugated globular protein with a molecular weight of around 64.4 Kd.
      1. This protein is 95% of the RBC’s dry weight or 33% of the RBC’s weight by volume.
         1. Around 65% of the Hb synthesis occurs during the nucleated stage of RBC maturation, and 35% occurs during the reticulocyte stage.
      2. Hemoglobin genes are present on chromosomes number 16 and 11.
         

         Hemoglobin genes location and normal structure

         1. Normal Hemoglobin consists of 2 α- chains and 2- β chains with four protoporphyrin rings with Fe⁺⁺ (ferrous).
            

            Hemoglobin normal structure

      3. Hemoglobin is the main oxygen carrier to the cells and the tissue.
      4. This also takes CO2 back to the lung.
3. The energy pathways for RBCs metabolism.
   1. Active RBC metabolic pathways are important for the production of an adequate amount of ATP level, which is necessary for:
      1. Hemoglobin function.
      2. RBC membrane integrity and change in shape.
      3. An adequate amount of reduced pyridine nucleotides.
      4. Maintaining the RBC volume.
   2. RBC generates energy almost mainly from the anaerobic breakdown of glucose.
   3. Mature RBC has a limited ability to metabolize fats and amino acids.
      1. RBC ATP needs  90% is generated by the Embeden-Meyerhof glycolytic pathway.
      2. 5 to 10% of the energy is provided by the metabolism of glucose – hexose monophosphate shunt.
         

         Red blood cell energy metabolism

4. RBC life history:
   1. RBCs from the front side are round but, when seen from the side view, look biconcave.
   2. This can be given the example of doughnut-shaped with depression in the center.
      

      Red blood cell morphology and biconcave shape

   3. It measures 7 to 8 µm in diameter.
   4. RBCs mature in the bone marrow, and when it comes to the peripheral blood, it takes 3 to 5 days.
   5.  The RBCs live in the peripheral blood for around 120 days. After completing their life, these are cleared in the spleen, liver, and bone marrow.
      

      Various forms of red blood cells

3. Functions of the red blood cells:
   1. The RBCs’ main function is to carry oxygen from the lung (arterial blood)  to the cells and the tissues. The oxygen is carried with a chemical combination with the hemoglobin.
   2. At the tissue level, oxygen is exchanged with the CO2 and brings CO2 in venous blood to the lungs.
   3. Normally O2 exchange occurs between 95% saturation in arterial blood with a mean arterial O2 tension of 95 mmHg and 70% saturation in venous blood with a mean venous O2 tension of 40 mmHg.

1. 4. The RBCs have to be in close contact with the tissue and successful gaseous exchange to carry hemoglobin. RBCs must be able to pass through the microcirculation.
   5. RBC traveling in the microcirculation in 120 days which is roughly 300 miles (480 Km).
   6. To fulfill all these above functions, the RBCs are flexible and biconcave discs.

Peripheral blood smears give a lot of information about the RBCs morphology and effects of various drugs or different types of anemias.

1. Anemia workup includes:
   1. Hb concentration.
   2. Hematocrit (Hct).
   3. RBC indices.
   4. Reticulocytes count.
   5. Evaluation of the peripheral blood smear.
   6. Bone marrow can also be advised to classify the anemia.
2. Defects of the RBCs formation and reason for anemia (Etiology):
   1. Deficiency diseases.
   2. Refractory anemia due to ineffective erythropoiesis.
   3. Defect of the bone marrow due to hypoproliferative anemias.
   4. Excessive loss of the blood like:
      1. Hemolysis.
      2. Hemorrhage.
3. Disorders of the RBCs leading to various types of anemias:
   1. Iron-deficiency anemia.
   2. Sideroblastic anemia.
   3. Anemia of chronic diseases.
   4. Thalassemia syndrome.
   5. Pernicious anemia.
   6. Folic acid deficiency anemia.
   7. Aplastic anemia.
   8. Pure red cell aplasia.
   9. Refractory anemia.
   10. Paroxysmal nocturnal hemoglobinuria.
   11. Hemolytic anemias.
   12. Hemorrhagic anemia.
   13. Hereditary spherocytosis.
   14. Hereditary elliptocytosis.
   15. Glucose-6-phosphate dehydrogenase deficiency.
   16. Pyruvate kinase deficiency.
   17. Sickle cell anemia and disease.
   18. Autoimmune hemolytic anemia.

Red blood cells assessment is based on:

1. RBCs volume:
   1. Normal MCV RBCs are called normocytic.
   2. High MCV RBCs are called macrocytic.
   3. Low MCV RBCs are called microcytic.
2. Hemoglobin contents:
   1. Normal Hb and MCHC are called normochromic.
   2. High MCHC RBCs are called hyperchromic.
   3. Low MCHC RBCs are called hypochromic.

Red blood cell indices are:

Clinical presentation	MCV fl	MCH pg	MCHC %
Normal	80 to 100	26 to 32	32 to 36
Normocytic anemia	82 to 92	25 to 30	32 to 36
Microcytic anemia	50 to 80	12 to 25	25 to 30
Macrocytic anemia	95 to 150	30 to 50	32 to 36

Microscopic evaluation:

Microscopic examination of the peripheral blood smears give information about:

1. The size (anisocytosis).
2. The shape (poikilocytosis).
3. The color (hypochromic or hyperchromic).
4. Intracellular inclusions.
5. Microscopic examination:
   1. First scan under the low power 10x and note:
      1. Staining quality of the smear.
      2. Also, check the center and edges of the slide to see there is no clumping of RBCs, WBCs, and platelets for the good distribution of the cells on the smear.
      3. Scan for the abnormal cells.
      4. Find the area where the RBCs should not touch each other.
      5. The RBCs should have a gradual central pallor.
   2. Scan the smear in high power 40x and note:
      1. WBCs abnormality.
      2. Can estimate WBCs count.
      3. Evaluate the RBCs morphology.
   3. Scan the smear in oil immersion 100x and note:
      1. Evaluate the RBCs anisocytosis, poikilocytosis, hypochromasia, polychromasia, and inclusion.
      2. Can evaluate the platelets count.
      3. WBCs differential can be done.

The following table explains the RBCs morphology changes and interpretation:

Terminology	Morphology	Explanation	Interpretation (Causes)
Microcytes		RBC size is smaller than normal	Iron Deficiency Anemia Thalassemia Hemoglobinopathy
Macrocytes		RBC size is larger than normal	Megaloblastic Anemia  Hypothyroidism Liver disease Alcoholism
Anisocytosis		Variation in RBC size	Seen in various types of anemia
Poikilocytosis		Variation in RBC shapes	Seen in various types of anemia
Polychromasia		RBCs are large and basophilic.	Seen with hemolytic anemia
Spherocytes		RBCs are small and round and no central pale area.	Seen in: Acquired autoimmune anemia Hereditary  spherocytosis
Elliptocytes		RBCs are rod-shaped (oval or elliptical)	Hereditary elliptocytosis
Target cells		RBCs have a darkly central area.	Thalassemia Iron deficiency anemia Liver diseases Post-splenectomy Hemoglobinopathy
Tear-drop poikilocyte		RBC looks like tears	Myelofibrosis Extramedullary hematopoiesis
Sickle cell	Sickle cell	Moon shaped RBC	Sickle cell anemia
Acanthocytes		RBCs have spikes, irregular projections	liver diseases Renal failure Abetalipoproteinemia
Schistocytes		RBCs show fragments and a variety of shapes and sizes.	DIC Prosthetic heart surgery. Microangiopathy Burns
Pencil cell		Elongated RBC	Iron-deficiency
Burr cells		RBCs are created and irregular.	Seen in Renal failure, Hypothyroidism, and liver diseases
Howell-Jolly bodies		RBCs show nuclear remnants.	After splenectomy Hypothyroidism
Heinz bodies		RBCs show denatured hemoglobin.	Seen in G6PD deficiency
Toxic granulation		Increased granules in polys	Indicate bacterial infection
Rouleaux formation		RBCs show clumping or aggregation.	Seen in Multiple myeloma or inflammation
Agglutination		RBCs are clumped	Seen in cold agglutinin

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