Reticulocyte Count (Retic count), and Interpretations
Sample for Reticulocyte Count
- EDTA blood is the best sample.
- Collect 5 to 7 mL of venous blood in the lavender-top tube.
- No fasting sample is needed.
Indications for reticulocyte count
- Reticulocyte count differentiates anemia caused by bone marrow failure, hemorrhage, or hemolysis.
- To check the effectiveness of Pernicious anemia, folate, and iron deficiency treatment.
- To assess the recovery of bone marrow function in aplastic anemia.
- To find out the effect of the radioactive material on exposed workers.
- Check bone marrow function in chemotherapy, radiation, and bone marrow transplantation patients.
Precautions for reticulocyte count
- Avoid counting insufficient numbers of RBCs and reticulocytes.
- RBC inclusions may be mistaken for reticulocytes like Pappenheimer, basophilic stippling, or Heinz bodies.
- Howell-jolly bodies may be mistaken as reticulocytes. Even the machine can count these as reticulocytes and give a false count.
- Pregnancy may cause an increase in the reticulocyte count.
Definition of reticulocytes:
- Reticulocytes are present in between the nucleated RBCs and mature peripheral blood RBCs.
- The reticulocytes are young, immature, nonnucleated RBCs.
- Reticulocytes are nonnucleated, fully hemoglobinized RBCs.
- The reticulocytes contain the reticular material, which is basically RNA, and it stains grey-blue (Aggregates of ribosomal RNA) with supravital stains like new methylene blue or brilliant cresyl blue.
- This is the microsomal and ribosomal material left in the RBCs.
- These large RBCs containing ribosomal RNA material look like reticulofilamentous material giving the name reticulocytes.
- The role of RNA is to synthesize hemoglobin, which is needed by mature RBCs for oxygen transport.
Reticulocytes maturation in the bone marrow:
- Reticulocytes lose the reticulum 24 to 27 hours after entering the blood circulation.
- But the premature stress reticulocytes have increased reticulum and need 2 to 2.5 days to lose their reticulum, resulting in longer peripheral blood maturation time.
- When Hb is low, the maturation time is longer, allowing more Hb production by the reticulocytes’ ribosomal RNA.
- RNA is precipitated as a dye-protein complex.
Reticulocyte activity may be expressed as:
- Absolute count.
- Reticulocyte production index.
- Reticulocyte %.
Maturation time is correlated inversely with hematocrit.
|Hct %||Maturation time of reticulocytes (days)|
- Increased reticulocytes indicate that bone marrow releases increased RBCs into the peripheral blood.
- Reticulocytes reflect replacing the daily RBCs destroyed each day, which is 1% of the red cell mass.
- Normal or low reticulocyte counts in anemia indicate inadequate bone marrow response or some anemia.
- Increased reticulocyte count indicates an overproduction of RBCs that may compensate for hemolysis or hemorrhage.
Life of reticulocytes in the peripheral blood and bone marrow response:
- When RBCs are released into the peripheral blood, they contain reticular material, which remains 1 to 2 days before the cell fully matures.
- RBC average life span = 100 to 120 days in the peripheral blood.
- Reticulocytes’ average life span = 1.0 days in the peripheral blood.
- Bone marrow replaces approximately 1% of the adult RBC every day.
- Another source:
- Reticulocytes circulate in the peripheral blood is 1 to 2 days.
- 0.5 to 2.5% are the normal reticulocytes in the peripheral blood. This indicates normal bone marrow activity when hemoglobin is normal.
- RBC’s mature form circulates in the peripheral blood for 120 days.
- When hemoglobin is low, and reticulocytes are 0.5 to 2.5%, it will indicate that anemia’s response is inadequate. This will be due to defective or decreased bone marrow production or a decreased amount of erythropoietin.
- In the case of anemia, hemoglobin will fall, the circulating RBC number will fall, and the reticulocyte number will increase.
Reticulocytes and bone marrow response:
|Reticulocytes count and Hb||Bone marrow activity|
Reticulocytes’ response to blood loss:
- The reticulocyte is the single most useful test to detect the hemolytic state.
- Reticulocytosis in a single episode of blood loss starts after 2 to 4 days, and the peak level is between 4 to 7 days. When hemoglobin becomes normal, then reticulocyte counts also become normal.
- In case of persistent reticulocytosis or a second rise in the reticulocyte count indicates continuing or second episode of blood loss. In iron deficiency anemia or anemia due to blood loss, the reticulocytes will become normal in 4 to 7 days when the iron supplement is given.
Reticulocyte count importance (functions):
- Bone marrow function.
- Elevated reticulocytes in the presence of normal hemoglobin indicate erythrocyte activity.
- Anemia typing and diagnosis can be made on reticulocytes count.
- >3% reticulocytes indicate hemolytic anemia, indicating compensatory bone marrow response.
- <2% will be seen in:
- Hypoproliferative disorders.
- Maturation abnormalities like microcytic or macrocytic anemia.
Methods to stain Reticulocytes
- Reticulocytes are not easily visible with the Wright or Giemsa stain.
- These are recognized as immature red blood cells in the peripheral blood smear.
- Reticulocytes need special vital stains like new methylene blue or cresyl blue stains.
- Reticulocytes are larger than normal RBCs and may be recognized as macrocytes. When present in sufficient numbers, it may give rise to an increase in MCV.
- In contrast to red-orange RBCs, reticulocytes in Wright’s stains look gray or blue-gray; this condition is called polychromatophilia.
New Methylene blue stain (NMB) for reticulocytes count.
- This new methylene blue stain is more specific for the reticulocytes.
- The staining visualizes reticulocytes with vital dyes like methylene blue that precipitate the RNA and organelles, forming a reticulum network.
New methylene blue (NMB) stain preparation:
- New methylene blue = 0.50 gram
- Sodium chloride = 0.70 gram
- Sodium oxalate =0.13 gram
- Deionized water = 100 mL
- Mix these three reagents for at least 15 minutes, filter them, and store them at room temperature.
- Filter before use all the time.
The procedure of NMB stain (reticulocytes stain):
- Add two drops of blood to three drops of NMB solution.
- If Hb is low, then add one more drop of blood.
- Mix and leave it for 15 minutes.
- Incubate the mixture for 10 minutes at 37 °C.
- Resuspends the cells before making the slides.
- Now make three thin smears and let them dry.
- Count a total of 1000 RBCs under the 100 x oil immersion lens.
- Try to make a thin smear with around 100 cells per field.
- Keep a record of reticulocytes as well.
- Reticulocytes appear greenish-blue and have different concentrations of reticulum formation.
- Count the reticulocytes on all three slides and compare the distribution.
Calculation of reticulocytes:
- Count RBCs and reticulocytes as well.
- If RBCs are, 1000 and reticulocytes are 8.
- Example : Reticulocytes = 8/1000 x 100 = 0.8%
Special stain with brilliant cresyl blue:
- Mix the blood with this stain and incubate for 15 to 30 minutes.
- Now make the smear from this blood sample.
- Count 5oo cells and calculate the percentage of reticulocytes out of 500 cells.
Reporting of reticulocytes:
- The reticulocytes are reported as the percentage of total RBCs counted.
- Reticulocytes % = Count 1000 RBCs and the number of reticulocytes.
- In the case of decreased reticulocytes, count 2000 RBCs.
- When 1000 RBCs are counted, the following shorthand formula may be used:
- % Reticulocytes = No. of reticulocytes counted/1000 RBCs.
Absolute Reticulocyte count:
- It is reticulocyte numbers in 1 mm3 of whole blood. This is not % of the RBCs.
- It has more value than the % reticulocytes.
- The normal range = is 24 to 84 x 109/L. (another reference is 25 to 75 x 109/L).
- Another reference to calculate absolute reticulocyte count:
Corrected reticulocyte count:
- In the state of anemia, reticulocyte % is not a true reflection of reticulocyte production.
- A correction factor must not be applied to overestimate marrow production because each reticulocyte is released into whole blood containing few RBCs, which means low Hct, thus increasing the percentage.
- The formula calculates the corrected reticulocyte count.
Reticulocyte production index (RPI):
- The peripheral smear should be carefully reviewed for polychromatic macrocytes, which can indicate stress reticulocytes.
- It needs correction for both RBCs count and the presence of stress reticulocytes. This value obtained is called the Reticulocytes production index.
- RPI corrects the hematocrit to a normal value of 45% and considers the reticulocytes’ maturation time at a particular hematocrit.
- At 1.0 day = 45% Hct.
- At 1.5 days = 35%. Hct.
- At 2 day = 25%. Hct.
- At 2.5 day = 15%. Hct.An RPI of >2.5 to 3.0 is generally indicative of hemolytic anemia.
|Newborn||3 to 7|
|0ne day||3 to 7|
|3 days||1 to 3|
|7 days||0 to 1|
|one month||0.2 to 2.0|
|1.5 month||0.3 to 3.5|
|2 month||0.4 to 4.8|
|2.5 month||0.3 to 4.2|
|3 month||0.3 to 3.6|
|4 to 12 month||0.2 to 2.8|
|Adult||0.5 to 1.5|
- Adult/elder/child = 0.5 to 2%
- Infants = 0.5 to 3.1%
- Newborn = 2.5 to 6.5%
- Reticulocyte index = 1.0
|Age group||% reticulocytes|
|Newborns||3% to 6% of total RBCs counted (Drops to adult levels in 1 to 2 months)|
|Adult||0.5% to 1.5% (Females may have a slightly higher %)|
|Reticulocyte index (RI)||1% corrected reticulocyte count|
|Hct correction for anemia = RI||Reticulocyte count x (patients’s Hct/45 x 1/1.85)|
- Adult = 0.5 to 2 % of the RBCs
- 24 to 100 x 109 reticulocytes/L.
- Newborn = 3 to 7%
- One week = 1.8 to 4.6%
- One month = 0.1 to 1.7%
- 6 months = 0.7 to 2.3%
- >6 months = 0.5 to 1.0%
- Adult = 0.5 to 1.5 % of total RBCs counted.
- Newborn = 3% to 6 %.
Increased reticulocyte values are seen in the following:
- Hemolytic anemia:
- Immune hemolytic anemia.
- Sickle cell disease.
- Primary RBC membrane defect.
- Hemolytic disease of the newborn.
- Deficiency of RBC enzyme.
- The bone marrow tries to compensate for RBCs’ shortened life and produces many RBCs, some of which are immature reticulocytes.
- The Post hemorrhage Increased occurs after 3 to 4 days. This indicates that bone marrow is trying to compensate for blood loss. There is a possibility of immature RBCs, which are reticulocytes.
- After treating anemia, in the case of deficiency anemia, the marrow responds by increased RBCs reticulocyte production when treatment is given, like iron, vitamin B12, or folic acid.
- An increased retics may be used as an index of the effectiveness of the treatment.
- After an adequate dose of iron in iron-deficiency anemia, the reticulocyte increase may exceed 20%.
- There is also an increase in the retics when pernicious anemia is treated by blood transfusion or vitamin B12.
Decreased reticulocytes values are seen in the following:
- Aplastic anemia.
- Anemia of chronic disease.
- Untreated iron deficiency and pernicious anemia.
- Radiation therapy.
- Bone Marrow infiltration by tumors leads to bone marrow failure.
- Myelodysplastic syndrome.
- Adrenocortical hypofunction.
- Anterior pituitary hypofunction.
- Chronic diseases.
- In these diseases, marrow production of the RBCs and reticulocytes is decreased.
What is the work of sodium chloride and sodium citrate in reticulate count staining
I searched to find the reference for sodium chloride and oxalate in the stain but could not find it. But I think their functions may be to stabilize the RBCs.