Performing a Reticulocyte Count

1. Performing a Reticulocyte Count

2. Reticulocytes • A reticulocyte is an immature RBC that still has remnants of RNA in the RBC’s cytoplasm. • A reticulocyte count gives the health care provider an idea of how many immature RBCs are in the patient’s peripheral blood. • This is only an estimate and is an indirect way of estimating the growth process of a person’s RBCs (erythropoiesis).

3. Reticulocytes • Red blood cells are produced in the bone marrow. • As they mature they loose their nucleus and move out into the peripheral blood circulation. • For the first 24 hours, while circulating they still contain the remnants of their RNA in their cytoplasm. • On a blood smear stained with Wright’s stain they appear larger and stain more bluish. • Stained with a supravital stain (new methylene blue or brilliant cresyl blue) will make the reticulocyte appear to have granules inside it. • Retics appear blue tinged with dark blue to purple granules or reticulum inside the RBC. • Retics are also larger than more mature RBCs. • Mature RBCs appear bluish green with no granules inside them.

4. Performing a Reticulocyte Count • The reticulocyte procedure is performed on capillary blood or venous blood collected in EDTA. • The reticulocyte count should be performed on blood within 4 hours of the collection time. • A small amount of well mixed blood is added to an equal amount of reticulocyte stain in a small vial. • The mixture is allowed to stand for 15 or so minutes depending on the type of stain used. • The mixture is then used to make a blood smear. • The smear should be allowed to air dry before the staining procedure begins.

5. Performing a Reticulocyte Count • There are two commonly used methods for counting reticulocytes. • Standard counting procedure • Miller Reticle procedure • Standard counting procedure • Dry smear is examined under oil immersion. • Total of 1,000 RBCs are counted. • Number of reticulocytes are recorded per 1,000 RBCs. • Retics are counted twice. • Once as a retic and a second time as an RBC.

6. Performing a Reticulocyte Count • Miller Reticle Procedure • Dry smear is examined under oil immersion. • A eyepiece with a Miller reticle can be used. • It is two squares. One large square with a smaller square in the corner. • For each field of view the retics are counted in the whole large square (including the small). • All RBCs lying within the small square are counted including reticulocytes and that number is recorded as red cells. • This is repeated until 100 to 200 RBCs are counted in the small squares.

7. Performing a Reticulocyte Count • A reticulocyte count is reported as the percentage of RBCs that are reticulocytes. • Each calculation depends on the counting procedure used. • Standard Calculating Procedure • # of reticulocytes counted X 100 = % Retics Total # RBCs counted • # of reticulocytes X 100 = % Retics 1000

8. Performing a Reticulocyte Count • Miller Reticle Calculating Procedure • The formula is different than the standard because of the Miller reticle squares used. • # of reticulocytes counted X 100 = % Retics Total # RBCs counted X 9

9. Reference Ranges • The reticulocyte percentage depends on age. • Newborns have a high reticulocyte count that decreases after 2 weeks of age. • In a healthy adult the retic count is usually stable. • About 1% of RBCs will stain as reticulocytes when using the supravital stains previously discussed. • Newborns 2.5-6.5% • Adults 0.5-1.5%

10. Reference Ranges • The increased reticulocyte percentage can be caused by….. • Hemolytic anemias • Hemorrhage • Heavy smoking • Living at high altitudes • Treated folate, iron, B12 deficiency • A decrease reticulocyte percentage can be caused by….. • Aplastic anemia • Bone marrow failure • Untreated Vitamin B12 deficiency and Iron deficiency