Unit III

Reticulocyte Count

3.1 Supravital stain and reticulocyte counting-Introduction.

3.2 Principle and procedure of staining and calculation

Reference values and interpretation.

Reticulocyte count

Reticulocytes are immature red blood cells released by the bone marrow into the bloodstream. Reticulocytes have remnant ribosomal RNA. RNA develops a net-like (reticular) appearance in the immature red blood cells. Thus it is called a reticulocyte. Reticulocyte counts are a measure of the bone marrow's ability to produce red blood cells. Reticulocyte counts help to distinguish different types of anemia. It also helps to identify the causes of anemia.

Supravital stain

The Supravital means “above life” or “beyond life”. Supravital stain is a technique to stain living cells without affecting their viability. In supravital stain, living cells are stained without their fixing or drying. Supravital stains penetrate living cells and selectively bind to the specific cellular components. It is most commonly used to stain reticulocytes.

The most commonly used supravital stain to stain reticulocytes is new methylene blue (NMB). NMB binds with RNA in reticulocytes. This develops a blue color in the reticulocytes. Thus these reticulocytes can be easily counted under a microscope. Other supravital stains are brilliant crystal blue and crystal violet. These dyes have also an affinity for remnant ribosomal RNA present in the reticulocytes. They develop specific colors in the reticulocytes.

Principle of Supravital staining: Supravital staining has the ability to penetrate living cells without causing significant damage or affecting the viability of the living cells. Supravital stain selectively binds with specific cellular components. This increases the visualization of living cells under a microscope.

The Supravital staining technique is used in hematology to count the number of reticulocytes in the blood sample. Supravital stains are vital dyes or fluorescent dyes that interact with remnant ribosomal ribonucleic acid (RNA) in reticulocytes. Examples are new methylene blue (NMB), brilliant crystal blue, and crystal violet. New methylene blue (NMB) is the most commonly used supravital stain. It binds with the remnant ribosomal ribonucleic acid (RNA) present in reticulocytes to develop blue color.

The procedure of Supravital staining technique for reticulocytes

The following steps are followed in the supravital staining technique for reticulocytes

1. Blood sample collection: Collect a small amount of blood from the vein in the arm by using a needle syringe or vacutainer system.

2. Blood smear preparation: Prepare a thin smear of blood sample on a clean dry glass slide. Allow it to dry in the air.

3. Staining: Flood the blood smear using supravital stain new methylene blue (NMB). Ensure the entire smear is covered. Allow stain solution to stain smear for 10 to 30 minutes. Stain enters the reticulocyte and binds with the remnant ribosomal ribonucleic acid (RNA).

4. Rinsing and drying: Rinse stained blood smears using distilled water or a suitable buffer solution. Rinsing removes the excess supravital stain. Allow the smear to dry in the air.

5. Microscopic examination: A stained smear is examined under low power objective (10X or 20X). Then it is examined under high power objective (100X or oil immersion).

6. Reticulocytes Count: Reticulocytes are counted in different fields under high power objectives.

7. Calculation: The number of reticulocytes counted is divided by the total number of RBCs.

Mix a small amount of the patient’s blood with the supravital stain. Incubate the mixture for a specified period. This allows stains to enter the reticulocytes. After incubation, prepare a blood smear on a clean sterile glass slide. Observe the smear under a microscope and count the number of reticulocytes.

Reference values of reticulocyte count

The reference value of reticulocyte count is expressed in the percentage or absolute count of reticulocytes about the total number of red blood cells.

• Reticulocyte percentage: The normal reference range of reticulocyte percentage is about 0.5% to 2.5% of total red blood cells.

• Absolute reticulocyte count: The normal reference range of absolute reticulocyte count is usually between 25,000 and 75,000 reticulocytes per microlitre of blood.

Interpretation of reticulocyte count

Reticulocyte provides valuable information about bone marrow functioning. Interpretation of reticulocyte count

1. Normal reticulocyte count: It indicates healthy bone marrow. Bone marrow produces red blood cells as per demand of the body

2. Increased reticulocyte count (Reticulocytosis): Increased reticulocyte count suggests hyperactive bone marrow. It occurs under the following conditions:

   • Hemolytic anemia: Increase in destruction of RBC stimulates bone marrow. Bone marrow produces more reticulocytes to compensate for RBC loss.

   • Acute blood loss: Rapid blood loss stimulates bone marrow to produce more RBCs. This ends up producing more reticulocytes

   • Recovery phase or iron deficiency: Iron deficiency decreases RBC number in blood. This stimulates the bone marrow to produce more RBCs. This ends up producing more reticulocytes.

3. Decreased reticulocyte count (Reticulocxytopenia): It indicates reduced production of RBCs from bone marrow. It occurs under the following conditions

   • Bone marrow suppression: Some medicines, toxins, radiation, or aplastic anemia suppress the functioning of bone marrow. This decreases RBC production.

   • Deficiency of erythropoietin: Erythropoietin is a hormone that stimulates RBC production in bone marrow. Chronic kidney disease decreases erythropoietin levels.

   • Nutritional deficiency: Inadequate intake of nutrient like iron, vitamin B12, etc. reduce RBC production.

Dr Pramila Singh

Variations in Physiological Values such as Hb, PCV, T.L.C., and Platelet count

Variation in Physiological values of hemoglobin

Several factors affect the physiological values of hemoglobin. These factors either increase or decrease the hemoglobin value.

1. Age: Haemoglobin values in Newborn is generally on the higher side i.e. 14 to 24 gram per decilitre. Hemoglobin level decreases during the first 6 months of life. The normal hemoglobin level in males is 12 to 16 grams per decilitre gm/dL. In women is 12 to 15 gm/dL.

2. Sex: Haemoglobin level in women is slightly lower than hemoglobin level in men. It is mainly due to menstruation in women and low muscle mass in women.

3. Altitude: Hemoglobin level in an individual is higher at a higher altitude than an individual at the sea level. High altitude has low oxygen levels in the air. The body increases blood hemoglobin levels to fulfill the body's requirement for oxygen.

4. Medical conditions: Various medical conditions either decrease or increase the hemoglobin level in blood. A decrease in hemoglobin level is called anemia and an increase in hemoglobin level in blood is called polycythemia.

Variation in Physiological values of PCV

Packed cell volume (PCV) is the volume of red blood cells in a given whole blood volume. The normal PCV range in adult males is between 40% and 54% and in adult females is between 37% and 47%. These values may vary slightly depending upon laboratories and health care providers. Some factors such as age, sex, and health status affect it. Variation in PCV values indicates medical conditions or factors affecting blood cell production.

1. Anemia: Low PCV values indicate anemia. Anemia may be due to blood loss, bone marrow depression, nutritional deficiencies, and some chronic diseases.

2. Dehydration: Dehydration causes a decrease in plasma volume. This led to an increase in PCV. It is not due to an increase in red blood cell production but due to a decrease in plasma volume.

3. Polycythemia: An abnormal increase in the number of red blood cells at normal value is called polycythemia. It may be due to bone marrow disorder, lung disease, or high altitude. In these conditions, the PCV value increases above the normal value.

4. Pregnancy: PCV value decreases during pregnancy. It is mainly due to the expansion of plasma volume during pregnancy.

5. Smoking: PCV value increases in smokers. It is due to nicotine's effect on red blood cells and plasma viscosity.

Variation in Physiological values of TLC

Total leucocyte count (T.L.C.) is the total number of white blood cells present in the given sample of blood volume. The normal range of T.L.C. is between 4000 and 11000 white blood cells per microliter of blood. The normal range of T.L.C. depends upon various factors such as age, sex, medical conditions, and laboratory techniques to measure T.L.C.

1. Infection: Microbial infection increases the T.L.C. It is due to the mobilization of leucocytes to fight microbial infection. It is characterized by an increase in the number of neutrophils, a type of leucocyte (WBC).

2. Inflammatory conditions: Certain inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, etc increase the T.L.C.

3. Stress and physical exertion: Intense physical activity, intense physical exercise, or intense emotional stress cause a temporary increase in T.L.C.

4. Medication: Certain medications such as steroidal drugs, and antibiotics affect WBC production and its function. This causes a change in T.L.C.

5. Bone marrow depression: Any condition that affects bone marrow activity changes T.L.C. Example: Leukemia. It causes an abnormal increase in WBC production.

6. Certain diseases: Cancer, autoimmune disorders, chronic allergic reactions, etc increase T.L.C.

Variation in Physiological values of Platelet counts

Normal platelet count in adults is between 150000 and 450000 platelets per microliter of blood. A Platelet count below this range is called thrombocytopenia. A platelet count above this range is called thrombocytosis. Platelet counts depend upon several factors such as age, sex, health conditions, medications, and also upon medical laboratories and countries.

1. Infection: Microbial infection temporarily decreases the platelet counts.

2. Autoimmune disorder: Autoimmune disorder attacks platelets. This decreases the platelet count.

3. Bone marrow disorders: Any disease that depresses bone marrow decreases platelet production. Such as aplastic anemia.

4. Medication: Chemotherapeutic agents and heparin decrease the platelet count.

5. Enlarged spleen: Any condition that enlarges the spleen decreases platelet count. Enlarged spleen trap platelets that lead to decreased platelet count.

Dr Pramila Singh