Reticulocytes are immature non-nucleated red cells that contain ribonucleic acid and ribosomes to synthesize hemoglobin after the loss of the nucleus.
After 2 to 2 1/2 days in the bone marrow, the reticulocyte is released into the vascular sinuses of the marrow. Then the reticulocyte maturation continues in the peripheral blood for another one day
Approximately 65% of the cells hemoglobin is made during the normoblast stage. The remaining 35% of cellular hemoglobin is made during the reticulocyte stage
The residual RNA in reticulocytes gives bluish tinge with Wrights stain and the cell is described as polychromatophilic erythrocytes. The polychromatophilia is due to the presence of basophilic ribosomes.
What is supravital stain
Supravital stains are used for coloring RNA in living cells in vitro.
Supravital stain such as Methylene Blue, Brilliant Cresyl blue are used for staining reticulocytes
Principle of reticulocyte staining:
The stain causes ribosomal and residual RNA to precipitate in living young RBC’s as Dye-ribonucleoprotein complex which aggregates into a reticulum and precipitate as deep bluish purple inclusions which appear as blue filamentous or granular material
The younger reticulocytes has more filamentous material while old reticulocytes have dot like inclusions.
The enumeration of reticulocyte count will be the measure of the number of cells delivered by the marrow to the blood each day. That is the measure of effective erythropoeisis
Normal reticulocyte count
Normal adult – 0.5% – 1.5% (25 – 75 X109/L)
New borns – 2.5% – 6.5% (this falls to adult range by the end of second week of life)
Reticulocyte staining procedure
Reagent –
1% of New methylene blue in a diluents of Citrate /Saline
New Methylene blue/ Brilliant Cresyl blue – 1.0 gm
3% sodium citrate – 20 ml (functions as anticoagulant)
0.9% Sodium chloride solution – 80 ml (Provides iso osmalality as that of blood)
Procedure –
3 drops of each reagent and blood are mixed in a test tube, incubated for 15 minutes at room temperature (370c) and remixed.
Two wedge films are made on glass slides and air dried.
Viewed microscopically with an oil immersion lens
Reticulocytes are pale blue with dark blue reticular or granular material and red cells stain pale blue
Reticulocytes should be calculated for 1000 RBC’s
Method using the Miller disc
Use a 100x objective and a 10x ocular secured with a Miller disc.
The Miller disc imposes two squares (one 9 times the area of the other) onto the field of view.
Find a suitable area of the smear. A good area will show 3-10 RBCs in the smaller square of the Miller disc.
Count the reticulocytes within the entire large square including those that are touching the lines on the left and bottom of the ruled area. Count RBCs in the smaller square whether they contain stained RNA or not. A retic in the smaller square should be counted as an RBC and a retic. Record RBC counted and retic counted separately.
Continue counting until a minimum of 111 RBCs have been observed (usually 15-20 fields). This would correspond to 999 RBCs counted with the standard procedure.
Reticulocyte (%) = total reticulocytes in square A X 100
Total RBC’s in square B X 9
What is the specific eye peice used in counting reticulocyte
Ehrlilchs eye peice- which is provided with a diaphragm having a square opening, the size of which is regulated by a small lever.
Absolute reticulocyte count
is the actual number of reticulocytes in 1L of whole blood. This is calculated by multiplying the reticulocyte % by the erythrocyte count.
Normal absolute reticulocyte count is approximately 50 X 109 /L
To avoid the overestimate of RBC production correction factor is used that is based on estimated maturation time of reticulocytes in the blood.
Corrected reticulocyte count –
Calculated based on normal hematocrit.
In anemia, a more accurate index of erythropoiesis is needed, than the relative reticulocyte count.
In anemia if the reticulocyte count is 1% in which even the number of RBC’s are less, it is not normal
For ex – absolute reticulocyte count – 0.01X 5 X 1012 = 50 X109/L
But in anemia – 0.01 X 2X1012= 20X109/L . So the person is lacking the production of 30X109/L
The corrected reticulocyte count overcomes this dilution problem and is useful in evaluating the bone marrow response in anemia.
The formula for corrected reticulocyte count uses the patients hematocrit in comparision to a normal hematocrit (45)
Patients hematocrit X reticulocyte count
45
In anemia where patients hematocrit is (19) reticulocyte count is
19 X1 = 0.4%
45
Reticulocyte production index –
An additional reticulocyte correction is needed if large polychromatophilic macrocytes are seen on the blood smear.
With increasing erythropoietin stimulation , the bone marrow releases reticulocytes before their normal 2 to 3 day marrow maturation period
This means the additional bone marrow maturation time is added to peripheral blood maturation time and it takes longer than the normal 1 day for the peripheral blood reticulocytes to become a mature erythrocyte
The more severe anemia, the earlier the reticulocyte is released. In a stimulated marrow, hematocrit levels are associated with early reticulocyte release and a prolongation of reticulocyte maturation in the peripheral blood i.e,
To correct for the prolongation of maturation of these circulating shift reticulocytes , the following formula is used,
Reticulocyte production index = corrected retic count in %
Maturation time in days
This correction is known as the reticulocyte production index (RPI) . RPI is a good indicator of the adequacy of the bone marrow response in anemia
RPI greater than 2 indicates an appropriate bone marrow response whereas RPI less than 2 indicates an inadequate compensatory bone marrow response.
By using reticulocyte stain the following also can be demonstrated
HbH inclusions seen like tiny multiple inclusions in the RBCs giving golf ball appearance seen in Thalassemia ( even the blood picture also will be microcytic hypochromic)
Heinz bodies – aggregates of denatured hemoglobin seen as single or multiple rounded inclusions seen in G6PD deficiency
High reticulocyte count ( reticulocyte count >1.5% and reticulocyte index > 3%)
Following therapy in Fe, folic acid, B12 deficiency
Low reticulocyte count (reticulocyte count <0.5%, and reticulocyte index<1%)
Aplastic anemia
Pure red cell aplasia
Fanconis anemia
Aplastic crisis due to Parvo virus
Bone marrow suppression in chemotherapy and in sepsis
Liver disease
Disordered RBC maturation
Iron deficiency anemia
Vitamin B12 deficiency
Folate deficiency
Anemia of chronic disease
Hypothyroidism
If blood that has been allowed to stand (unstained) for as long as 24 h count will tend to decrease after 6–8 h unless the blood is kept at 40c
Fluorescence Methods
Manually by fluorescence microscopy
Add 1 volume of Acridine orange solution (50 mg/100 ml of 9 g/l NaCl) to 1 volume of blood
Mix gently for 2 min
Make films on glass slides, dry rapidly and examine with a fluorescent microscope
RNA gives an orange–red fluorescence, whereas nuclear material (DNA) fluoresces yellow
The amount of fluorescence is proportional to the amount of RNA
Not suitable for routine use for reticulocyte counting
Automated reticulocyte counts
Fluorescent staining combined with flow cytometry
Auramine O or polymethine with oxazine (Sysmex)
Traditional New methylene blue (Beckman Coulter, Abbott)
other flourescent dyes used are- Thiazide orange, Thioflavine
Sources of error
Refractile appearance of erythrocytes should not be confused with reticulocytes
Filtration of stain is required as precipitated material is present which can resemble a reticulocyte
Erythrocyte inclusions can be confused for reticulocytes
Howell jolly bodies appear as one or two deep purple dense structures
Heinz bodies – stain as light blue –green and present at the edge of the erythrocyte
Pappenheimer bodies are most often confused with reticulocytes. These purple-staining iron deposits appear as several granules in a small cluster. If pappenheimer bodies are suspected stain with wrights stain to verify their presence
High glucose levels causes reticulocytes to stain poorly
Falsely decreased reticulocyte counts can result from under staining the blood with new methylene blue
References
Praful B. Godkar, Darshan P. Godkar.Textbook of medical laboratory technology 2007. Second Edition
Sabitri sanyal, Aparna Bhattacharya.Clinical pathology A practical manual 2017. Third edition.
By
Dr. V.Shanthi (Professor of Pathology, Narayana Medical College, Nellore)
Dr.Shyam Sundara Rao (Professor of Pathology, Narayana Medical College, Nellore)
