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Introduction of Reticulocyte

A reticulocyte is a young and immature red blood cell (RBC). Its number increases only when there is a reason for the body must produce extra red blood cells. This process can be measured by the reticulocyte count as shown above image. It is also known as Retic Count or Reticulocyte Percent or Reticulocyte Index or Corrected Reticulocyte and  Reticulocyte Production Index( RPI). Its count helps to determine the number and/or percentage of reticulocytes in the blood and is a reflection of recent bone marrow function or activity.

Determination of Reticulocyte count

Principle

The supravital staining method is used for reticulocyte count. Blood is mixed with the stain and the stain enters living conditions. The RNA in the cells is precipitated by staining as a dark blue network or reticulum. A blood smear is made afterward. Since a direct count is not possible, a relative count is taken against the number of RBCs and expressed as a percentage of red cells.

Test Requirements

• Clean and grease-free slides
• Test tubes (15x 125 mm)
• Dropper or Pasteur pipette with rubber teats
• Capillary tube
• Test tube rack
• Microscope
• Spuravital stain ( Brilliant cresyl blue or New methylene blue)
• Water bath or incubator

Specimen: EDTA or heparinized blood. A blood sample is collected by inserting a needle into a vein in your arm or sometimes from a finger stick or heel stick (infant). The specimen need not be a fasting sample. Perform the test within 2 to 3 hours of blood collection.

Procedure

1. Take a test tube.
2. Add two drops of blood and an equal amount of stain ( two drops) using separate pipettes or droppers and mix thoroughly.
3. Cover the tube with a cotton plug and keep it at 37°C for 30 minutes.
4. Prepare a thin smear of the stained blood specimen by using a spreader slide. Ary dry the smear.
5. First, examine the smear under the low power objective (10X)  and locate a portion of the smear, where the RBCs are evenly distributed.
6. Change to the oil immersion objective. Reticulocyte is identified by fine. deep violet and granules arranged in a network. Red blood cells pale blue. Simultaneously count reticulocytes and RBCs in about 15 fields.

Calculations

Reticulocyte %= (Number of reticulocytes counted X 100)/Number of RBCs counted

Foe example suppose,

Number of RBCs counted= 600

Number of reticulocytes counted= 30

Reticulocyte %= (30×100)/ 600

=5%

Clinical Significance  of Reiculocyte count

1. Red blood cells are produced in the bone marrow, where blood-forming (hematopoietic) stem cells differentiate and develop, eventually forming reticulocytes and finally becoming mature RBCs.
2. The number of reticulocytes in peripheral blood is a reflection of red blood cells forming the activity of bone marrow.
3. An increase in their number indicates increased activity of the bone marrow and this is called reticulocytosis. e.g. Hemolytic anemia, acute blood, polycythemia vera, and tumor that produces excess erythropoietin.
4. Repeated absence or low counts of reticulocytes indicate bone marrow suppression. e.g. aplastic anemia, Iron deficiency anemia, pernicious anemia or folic acid deficiency, radiation therapy, bone marrow failure caused by infection or cancer, severe kidney disease; this may cause a low level of erythropoietin, alcoholism, and endocrine disease.

Normal Range of Reticulocytes

Adult:0.2 to 2 %

Infant: 2 to 6%

Conditions of Ordering Reticulocyte Count

A reticulocyte count may be tested in the following conditions-

• When complete blood cell count (CBC) results show a decreased RBC count and or a decreased hemoglobin and hematocrit.
• When a clinician wants to evaluate bone marrow function.
• When you have signs and symptoms of anemia or chronic bleeding, such as paleness, lack of energy, fatigue, weakness, shortness of breath, and/or blood in the stool.
• When you have been diagnosed and are being treated for a condition known to affect red blood cells production, such as iron deficiency anemia, vitamin B12 or folate deficiency, or kidney disease (which can affect the production of erythropoietin, a hormone produced by the kidneys that stimulate RBC production by the bone marrow)
• When you are undergoing radiation or chemotherapy.
• When you have received a bone marrow transplant.
• Occasionally when you have an increased number of RBCs and elevated hemoglobin and hematocrit (HCT), to help determine the degree and rate of overproduction of red blood cells.

Keynotes

1. When you have anemia, the % of reticulocytes present in the blood may appear high compared to the overall number of red blood cells. In order to get a more accurate assessment of bone marrow function, a calculation known as a corrected reticulocyte count may be reported.
2. Corrected reticulocyte count (%) = reticulocyte (%) x (patient HCT/45)  ans 45 is considered the average normal hematocrit.

References

1. https://eclinpath.com/hematology/tests/reticulocyte-percentage/
2. https://www.ihtc.org/hereditaryspherocytosis/#:~:text=These%20young%2C%20immature%20red%20blood,measured%20by%20the%20reticulocyte%20count.https://labtestsonline.org/tests/reticulocytes
3. https://www.webmd.com/a-to-z-guides/reticulocyte-count-test#1
4. A Test Book of Medical Laboratory Technology-Praful B. Godaka and Darshan P. Godkar, 2nd Edition
5. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/reticulocyte
6. https://www.healthline.com/health/reticulocyte-count