A reticulocyte is a immature Erythrocyte that has lost its nucleus but retains aggregates of RNA within its ribosome. Mature RBC do not contain RNA.
DEVELOPMENT OF RBC
Earliest Erythrocyte identifiable is “Pronormoblast”. Also called RUBRIBLAST. It divide and give 16 mature RBC through 4 cell division within 72 hours. Cell size 12-25 µm. nucleus is almost 80% of the cell.
Next stage of RBC development. Clee size- 12-17 µm. nucleus occupy 75% volume of cell.
12-15 µm size, having round small nucleus-that may be eccentric. Heglobin starts to synthesize. Blue RNA mixed with haemoglobin gives the cytoplasm an opaque, gray-violet colour called polychromasia. As Hb synthesis increase, cell become more pinkish in colour.
Slightly smaller, 8-12 µm. cytoplasm become more pinkish. Nucleus is pyknotic or degenerated. Nucleus are exruded in this stage. Sometimes the nucleus is not completely extruded, some remnants remains- these are called “Howell-Jolly” bodies. These Howell-Jolly bodies are removed by spleen and not found in normal peripheral RBC. If any abnormality in RBC development occurs, the abnormal cells are removed by Macrophase in bone marrow, RBC are nor released in the circulation- called “Ineffective erythropoiesis”.
Slightly larger than mature RBC. After extrusion of nucleus, RBC remains in Bone Marrow for 2-3 days. Then they are released into circulation through marrow sinusoids. After release into peripheral circulation, they need 24 hours to mature fully. Reticulocytes contain 1. Golgi apparatus remnents 2. Residual of Mitochondria and 3. Residual RNA. Stain with methylene blue or brilliant cresyl blue cause RNA to precipitate or aggregate to form a visible network of strands or clumps under light microscopy. Tis RNA takes the dye in living states, so the technique is called “Supravital Staining”.
Cell size is 7.2 µm. surface areas or cell membrane is more than cytoplasmic contents- so cells retain biconcave shape, slightly oval. This helps RBC to travel through narrow capillaries changing its shape. Mature RBC does not contain nucleus or mitochondria, so they do not produce any proteins or haemoglobin.
HOW TO COUNT RETICULOCYTE
The RNA in ribosome inside RBC must be stained supravitally, ie, when RBC are in living state and not fixed. Ribosome have the property of reacting with certain basic dye, like
- Azure B
- Brilliant cresyl blue
- New methylene blue and form a blue or purple precipitate.
More immature the RBC, more the precipitation will be. More immature RBC shows coarse precipitate, more mature one shows only few dots. According to maturity, Reticulocytes can be divided into 4 group. 1. Most immature, 2. Intermediate group 3. Late stage of Intermediate group 4. Most mature Reticulocytes. More Reticulocytes are released into circulation in response to erythropoietic stimulation due to any cause.
STAINING of RETICULOCYTE:
EDTA mixed blood are commonly used for examination.
Staining solution: 1.0 gram of Mew methylene blue is dissolved in 100 ml of iso-osmotic phosphate buffer ph 6.5 or with isotonic saline. If EDTA blood is not used, sodium citrate 2.0 gram may be mixed with Buffer or isotonic normal saline as anticoagulant.
- Take 2-3 drop of dye in a 75x10mm test tube
- Add 2-4 volume of patient’s EDTA blood and mix.
- Keep the mixture at 37 degree centigrade for 15-20 min.
- Resuspend the blood by gentle mixing
- Make smears- air dry. Do not fix or counter stain.
See under light microscope in oil immersion.
- Exact volume of blood to be added depends upon the RBC count. Large amount should be given in anaemia and smaller proportion to be given in polycythemia.
- In a successful preparation, the reticulofilaments are stained deep blue and non reticulated cells stained diffusely shadesof pale greenisg blue.
- Smears should not be counterstained
- If unstained blood kept at 4 degree centigrade for 24 hours, it may give satisfactory count, but counts tend to fall after 6-8 hours .
STAGE OF MATURATION OF REYICULOCYTE
Considering the maturity, reticulocyte can be divided in to 4 group (Dacie)
- Group-I : most immature.
- Group-II: intermediate group.
- Group-III: Intermediate group
- Group-IV: most mature
Use the X100 oil immersion object and choose an area not too thin- just where RBC is not overlap with each other.
If Reticulocytes are less then to achieve accurate results, very large number of cells to be surveyed. If Reticulocytes is around 10%, then 100 reticulocytes to be counted.
Reticulocytes count is expressed as percentage of total RBC. Calculation is very simple.
Say total Reticulocytes is “x” in 100 fields, and total RBC number is “Y”. then the calculation is =X/Y*100. This will give percentage of Reticulocytes.
If “X” is 75, “Y” is 3000, then X/Y = 75/3000= 0.025 x100=2.5%.
Absolute Reticulocyte count is: ( % of Reticulocyte x total RBC count)/100
Example: if RBC count is 4.0 million/cu.mm, Reticulocyte percentage is 2.5%, then Absolute Reticulocyte count will be : 4000000×2.5/100= 100000/ cu.mm.
RETICULOCYTE COUNT BY FLUORESCENT MICROSCOPE
Reticulocyte count by fluorescent microscopey gives better results.
Principle: Acridine orange dye gives RNA orange-red fluorescence, while DNA fluorescence is yellow.
Preperation: Mix 1 volume of blood with 1 volume of Acridine orange solution (50mg /100 ml isotonic Nacl) à mix gently for 2 minutes. Films are made on glass slides and examined under fluorescence microscope.
Acridine dye fade rapidly and more rapidly in light. So examine as soon as possible.
NORMAL RETICULOCYTE COUNT
- In case of newborn, it ranges from 2-6%
- Reticulocyte count in adult ranges from 0.5% -2%
- Absolute count : 20,000-48,000/ cu.mm of blood.
ABNORMAL RETICULOCYTE COUNT;
Reticulocyte count increases where increased RBC formation or erythropoiesis occurs. If RBC demand increases due to any cause, such as anaemia, Bone Marrow starts to increase RBC formation, and in demand premature RBC are released into circulation.
Reticulocyte count decreases in case of Bone Marrow suppression due to any cause.
Conditions where Reticulocyte counts increases:
- Haemolytic anaemia ( Thalassemia/ other cause)
- Pernicious anaemia
- Regenerative phase of anaemia
Conditions where Reticulocyte counts decreases:
- Hypoplastic anaemia, where bone marrow produce less number of blood cells.
- Myelofibrosis- where fibrosis occurs in Bone Marrow.
- Cytotoxic drugs
- Bone marrow suppression due to any cause.
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